Characterization of the NIPBL Protein Associated with Cornelia De Lange Syndrome

Characterization of the NIPBL protein associated with Cornelia de

Lange Syndrome

A Thesis

Submitted to the Faculty

Drexel University

Daniel K. Keter

in partial fulfillment of the

requirements for the degree

Master of Science

June 2008

Acknowledgements

I would like to thank Dr. Mark S. Lechner for his advice, my committee members, Dr. Mary K. Howett for her time and invaluable assistance, Dr. Aleister Saunders and Dr. Felice Elefant. My regards also to Dr. Ian Krantz and Dr. Matthew Deardorff of the Childrens Hospital of Pennsylvania for their assistance, and my family members for being by my side.

iii

TABLE OF CONTENTS

List of Tables v

List of Illustrations vi

Abstract vii

Introduction 1

Cornelia de Lange syndrome 1

NIPBL and its relation to CDLS 3

NIPBL homologs and its chromosomal and DNA repair roles 7

Chromatin 17

Chromatin and its importance in gene regulation 18

Heterochromatin Protein 1 (HP1) 21

HP1 binds other proteins by its chromoshadow domain 22

HP1 in epigenetic silencing, gene regulation and chromatin remodeling 28

Malfunction/mutation of chromatin in other disease syndromes 29

Rationale, Hypothesis and Experimental Objective 30

Materials and Methods 32

Plasmid construction 32

Cell culture and transfection 32

Immunoflourescence Analysis 33

Doubling time 33

Western blot analysis 34

Antibodies 35

Recombinant proteins 35

Mouse protein heart extracts 35

Metaphase spreads 36 iv

Results 37

Characterization and morphological characteristics of CdLS cells 37

Specificity and colocalization of anti-NIPBL and anti-HP1 antibodies by 37 immunostaining

Characterization of the morphological characteristics of human fibroblast cells 40 with mutant and wild type NIPBL

Characterization of anti-NIPBL antibodies in cell lines by Western blot analysis 44

Analysis of precocious sister chromatid separation in Human and mouse embryo 49 Fibroblasts

Discussion 53

Future Experiments 57

List of References 60

LIST OF TABLES

Table 1: NIPBL homologs 9

Table 2: NIPBL belongs to a group of adherin proteins observed to have roles in 9 chromos chromosome adhesion

Table 3: Adherin and cohesion subunits are conserved 12

Table 4: Proteins shown to interact with the HP1 chromoshadow domain 24

Table 5: Morphological differences between mutant and wild type human fibroblast 41 CdLS cells

Table 6: Characterization of NIPBL nuclear staining morphology with rabbit anti-Scc2N 43 antisera

Table 7: Table showing the antibodies used to detect the NIPBL protein 48

LIST OF ILLUSTRATIONS

Figure 1: Some of the commonly observed CdLS symptoms from left to right, jointed 2 gggggggg eyebrow, hirsutism and oligodactyly.

Figure 2: Map of the delangin protein mapped along with locations of known NIPBL 6 mutations known to cause CdLS with protein interaction sites.

Figure 3: A model of how Scc2/Adherin facilitates chromosomal binding of cohesin 13 (S. cerevisiae)

Figure 4: A model of how NIPBL loads cohesin onto chromosomes 16

Figure 5: A model of heterochromatic self-maintenance by the SUVAR39H1/HP1 20 Complex

Figure 6: An illustration of the HP1 protein and its complex with NIPBL 25

Figure 7: Immunopurified FLAG-HP1α complexes from HEK293 nuclei 27

Figure 8: Colocalization of NIPBL and HP1 in U2OS cells 39

Figure 9: Immunofluorescent staining of CdLS and control human fibroblast cell lines 42

Figure 10: Western blots showing the expression of the NIPBL recombinant protein 46

Figure 11: Western blot of mouse heart extracts showing the specificities of 2 Nipbl 47 antibodies, affinity purified mNIPBL Ab 5.2.1 and rNIPBL Ab XSCC2N

Figure 12: Chromosome misalignment in ∆NIPBL and wild type human fibroblast cells 50

Figure 13: Chromosome misalignment in ∆NIPBL and wildtype mouse embryo fibroblast 51 Cells

Figure 14: An example of determination of chromosome misalignment in cells 52

vii

ABSTRACT

Characterization of the NIPBL protein associated with Cornelia de Lange Syndrome.

Daniel K. Keter Mark S. Lechner Ph.D

Cornelia de Lange Syndrome (CdLS) is a disorder that variably affects many physiologic

features of the human body. These include but are not limited to deformed limbs, facial features

and mental retardation. CdLS occurs in approximately 1 in 10,000 births. Of all CdLS cases,

50% have recently been associated with heterozygous mutation of the NIPBL gene. Four cases of

CdLS have been associated with mutation of SMCL1 which encodes a cohesin subunit. NIPBL is a homolog of the Drosophila melanogaster gene Nipped-B which has been shown to have roles in chromosome cohesion and gene regulation. Because mutant NIPBL as characterized in CdLS affects many organs in the body, we and others speculate that NIPBL has important roles during early stages of development. It has been shown that NIPBL binds to another protein, HP1. HP1 is known to be involved in chromatin based regulation by controlling access of transcription factors to the DNA strand. The proposition therefore would be that, since HP1 is a gene regulatory protein and NIPBL binds to it, a mutation on NIPBL would lead to an abnormal gene expression that leads to dysfunctional cell behavior displayed by Cornelia de Lange Syndrome.

The NIPBL protein has not yet been well characterized in human cells. The hypothesis would be that NIPBL has an important role in gene expression. When this role is disrupted, it leads to defects in gene expression such as those seen in CdLS. To test this hypothesis, we characterized the NIPBL protein properties. Using immunohistochemistry, we were able to show that NIPBL colocalizes with HP1 distinctly in the nucleus. Physical observation of ΔNIPBL cell lines showed lower doubling times and had a higher incidence of the ‘wrinkling’ phenomenon compared to wild type cell lines. At the protein level, only purified recombinant proteins were able to be viii detected by mouse monoclonal and rabbit polyclonal antibodies. Only one α-NIPBL antibody,

XScc2N was able to show a protein at 300kDa which corresponds to the size of NIPBL. It was also confirmed that a mutation of NIPBL leads to increased sister chromatid separation. The same did not hold true for mouse embryo fibroblasts. Characterization of the NIPBL protein would help better understand the resultant role of NIPBL in gene expression and cellular events that underlie the disease process in CdLS.

1. Introduction

General Background.

Cornelia de Lange syndrome (CDLS), sometimes called Brachmann-de Lange

syndrome, is characterized by multiple symptoms that include mental and growth

retardation, deformed limbs, and facial features etc (figure 1) [1, 3-5]. CdLS was first

described by Dr. W Brachmann in 1916[6]. It was later followed up by Dr. Cornelia de

Lange in 1933[6]. Since 1965, the syndrome was known as Cornelia de Lange syndrome

until Opitz and colleagues argued for Brachmann who had first documented its

occurrence hence the term Brachmann- de Lange syndrome[7]. It is estimated that CdLS occurs in approximately 1 in 10,000 people [6]. Numerous papers in different areas of

study have listed additional symptoms associated with this syndrome. [8-10]. Currently,

the disease process of CdLS is not well known and requires a lot of study into the

underlying molecular mechanisms accounting for the incidence of CdLS, its prevalence,

and the variation seen in the phenotypes of affected patients.

Figure 1: Some of the commonly observed CdLS symptoms from left to right, jointed eyebrow, hirsutism and oligodactyly. (Adapted from Krantz, I.D. et al 2004)

NIPBL and its relation to CDLS

Prior to the correlation of NIPBL mutation with CDLS, the NIPBL protein was shown to share a PxVxL binding motif with Heterochromatin protein 1 (HP1) [11].

Lechner and colleagues showed that HP1 and NIPBL physically interacted via the

chromoshadow domain (CD) of HP1 [12]. In June 2004, two groups identified that the

same gene, NIPBL, when mutated, caused CdLS.[5, 13]. Until then, there had been extensive research to identify candidate chromosomal locations for the CdLS gene.

Previously, the most probable region based on similarities between the 3q syndrome and

CdLS had been on region 3q26.3. [1, 14]. Then with collaboration and new techniques, the two groups, one headed by Dr. Ian Krantz at the Children’s Hospital of

Pennsylvania(CHOP) and the other headed by Dr. Tom Strachan at the University of

New Castle (UK) independently identified the NIPBL region to be on chromosome

5p13.1 after collaborative linkage analysis. Krantz et al used a linkage exclusion mapping approach. With fine mapping, Krantz et al managed to pinpoint the mutated chromosome at 5p13, a region not excluded by multipoint lod scoring analysis. In further support of the data, a child with a de novo translocation at 5p13.1-p14-2 narrowed down the probable site of the mutated gene in CdLS to 5p13 [5]. With mutational analysis,

Northern blots and cDNA analysis, they identified NIPBL as a 9.5kbp gene that coded for a protein that was 2804aa in size and containing 47 exons at chromosome 5p13.1 [5].

Tonkin et al took a different approach. Initially they used a high-density Bacterial

Artificial Chromosome (BAC) microarray comparative genome hybridization screen but could not find consistent patterns to link candidate chromosomal regions that linked to

CdLS [13]. However by chromosome breakpoint analysis, they identified the critical 4

region on 5p13.2 which was not excluded by multipoint scoring analysis among four

other sites [13]. The child with the de novo translocation at 5p13-5p14.2 also led them to characterize the 5p13.1 critical region. Through fluorescence in situ hybridization (FISH) mapping, they were able to identify the NIPBL gene.

The protein product of NIPBL is a protein named delangin (figure 2) [5, 13]. The

NIPBL mutation is heterozygous dominant [5]. No homozygous mutations of NIPBL have been discovered leading to speculation that it could be lethal in the embryonic stage.

Various mutations mapped on the NIPBL gene are predicted to cause a truncated protein or in a few cases an untranslated protein [5, 15]. In theory, the wildtype allele therefore is not able to produce enough of the functional NIPBL protein to allow normal function of the cell. A cell with the NIPBL mutation is unable to sustain normal development due to the haploinsufficient state hence evidenced by CdLS. It has been shown that there are significant differences between mutation positive and negative individuals in terms of growth rates, limb reduction and development, and cognitive abilities (figure2) [3].

Protein truncating mutations have been correlated with more severe symptoms [16, 17]

Most CdLS cases occur by spontaneous mutations. However, there are a few reported exceptional cases of mutations that are passed in the family [18]. To date, only

20-56% of the CdLS cases have been shown to have a mutation in NIPBL [5, 8, 16, 19].

The inability to completely identify all the causes of CdLS could be due to inadequate

detection techniques or that there may be other genes yet to be identified that also cause

CdLS or a closely related syndrome. Alternatively, a protein critical to cell pathways that

involves NIPBL may also be a mutation candidate for CDLS. Other deletions such as on

chromosome 3q have been linked to CdLS [20]. A case involving X-linked CdLS was 5 recently reported. It involved the mutation of the SMC1L1 protein in four male relatives owing to X-linked inheritance. [21] SMC1L1 is one of the subunits that make up the cohesion complex (see table 3) that hold together sister chromatids in a cell.

NLS

Figure 2: Map of the delangin protein with locations of known NIPBL mutations known to cause CdLS. Protein interaction sites are also shown. Using Vector NTI (Invitrogen, Carlsbad, CA), a sequence analysis software, the delangin protein was mapped along with known NIPBL mutations known to cause CdLS. The NIPBL protein is 2804 aa long. The dark green triangles are known mutation sites. PxVxL is a binding motif shared among proteins that bind to the epigenetic portion of HP1. The purple rectangle is CLR, a caldesmon like repeat while NLS is a nuclear localization signal. The light green boxes are HEAT(Huntington, Elongation Factor 3, PR65/A, TOR) repeats. Mutations sites were obtained from [3, 5, 13].

The full length NIPBL protein is 2804 amino acids long. Its sequence along with other proteins predicted to physically interact due to predicted motifs and domains were mapped onto the sequence to provide clues into the proteins function (fig. 2). A

Caldesmon like repeat shown as a rectangle spans residues 489-1025. Caldesmon (CDM) is a potential actomyosin regulatory protein found in smooth muscle and nonmuscle cells.

Domain mapping and physical studies suggest that CDM is an elongated molecule with an N-terminal myosin/calmodulin-binding domain and a C-terminal tropomyosin/actin/calmodulin-binding domain separated by a 40-nm-long central helix.

Caldesmon is associated with muscle contractility.[19, 22] The PxVxL motif is a binding motif that NIPBL shares with several proteins that bind Heterochromatin Protein 1 (HP1)

(See figure 6 and 7). The PxVxL motif is essential for recognition of the HP1 binding site.[12] NIPBL also has a nuclear localization signal. We speculate that NIPBL acts like a transport molecule which would be consistent with the prediction that it physically - interacts with the Caldesmon-like repeat. There are five HEAT repeats also mapped the

NIPBL protein. HEAT repeats are indicators of possible protein-protein interactions[12].

The HEAT repeats are conserved among species that have a homologous protein to

NIPBL. The NIPBL protein on figure 2, shows clustering of CdLS mutations around the sites of protein-protein interactions and around the nuclear localization signal (NLS). It is possible that NIPBL mutations disrupt normal protein-protein interactions and or transport to the nucleus.

NIPBL homologs have chromosomal and DNA repair roles.

The exact function of NIPBL remains unknown. NIPBL gene homologs in other organisms were identified and this gives clues into the function of the NIPBL gene. Long 8 before the NIPBL gene had been identified, its homologs in Drosophila melanogaster had been characterized in numerous studies. The D. melanogaster homolog, the Nipped-B gene, which when mutated was shown to cause nipped wings, was first characterized during a genetic screen to identify genes that regulate the cut and ubx. The cut gene encodes a homeobox protein that is required for proper cell specification and organogenesis of a number of tissues that in adult and drosophila larvae. The ubx gene on the other hand is responsible for the segmentation of the thorax in drosophila [23]. By using the conserved homology to this gene throughout higher organisms, NIPBL

(Nipped-B Like) was derived [5, 13]. Known homologies can be seen in Table 1.

Organism % Similarity to NIPBL M. musculus 92 R. norvegicus 86 D. melanogaster 26

G. Gallus 73

X. laevis 27 T. nigroviridis 36 A. gambiae 24 A. melliflora 23 C. elegans 23

A. nidulans 15

S. cerevisiae (Scc2) 14 S. pombe (mis4) 14 C. cinerus (Rad9) 17

Table 1: NIPBL homologs in other organisms were also identified. Human NIPBL has been shown to have substantial homology to other mammals. [1, 2]

Organism Adherin Demonstrated functions

C. cinereus Rad9 Meiotic DNA repair, chromatid cohesion, homolog pairing S. pombe Mis4 Mitotic sister chromatid cohesion, DNA repair S. cerevisiae Scc2 Mitotic sister chromatid cohesion D. Nipped-B Long-range gene melanogaster activation, sister chromatid cohesion X. laevis XScc2A,B Mitotic sister chromatid cohesion H. sapiens NIPBL- Clues from patient A,B symptoms suggest early physical and mental development

Table 2: NIPBL belongs to a group of adherin proteins observed to have roles in chromosome adhesion[1, 2] 10

NIPBL is homologous to proteins of other species. Table 1 shows percent sequence similarity. NIPBL fungal homologs have been well characterized. They include

Saccharomyces cerevisiae (Scc2), and Coprinus cinereus (Rad9)[24]. It has been previously shown that the genes Scc2, Mis 4 and Rad 9 are essential for sister chromatid cohesion. Separate studies listed them as essential for the loading of cohesin, an important molecule necessary for linking sister chromatids [25-28]. This group of proteins are called adherins because of their importance in sister chromatid cohesion

(Table 2). Frog XScc2 and human NIPBL have also been recently shown to be important in sister chromatid cohesion [29, 30].

During cell division, spindle fibers align the chromosomes along the middle of the cell nucleus. The paired chromosomes separate at the kinetochores and move to opposite sides of the cell. Equal distribution of chromosmes in daughter cells is essential for the life of the cell. Unequal distribution leads to cell cycle block, aneuploidy and cell death.

Defects in cell cycle events such as spindle formation have been known to cause cancer and birth defects. [31] The sister chromatids are held together right until the stage of metaphase by cohesin. Cohesin consists of four subunits that are conserved from yeast to humans. The subunits vary depending on the organism. It has also been shown that other than the cohesin subunits, there are other proteins that actively take part in the formation of cohesion (Table 3) [2].

Cohesin is important for the successful completion of the cell cycle. The cell goes through the G1, S, G2 and M-phase (Figure.3.). It is at the M phase that the cohesin is dissolved to allow separation of the sister chromatids into the daughter cells. At the S phase, sister chromatid cohesion is established. As a model of how sister chromatids are 11 joined together by cohesion, the cohesin subunits trap the DNA strand such that the replication fork can go through it. As a result of the replication, the newly synthesized

DNA strand is trapped.

S. S. C. D. Homo Sapiens cerevisiae pombe elegans Melanogaster Cohesin Smc1 Smc1 Psm1 Him-1 Smc1 Smc1/Smc1L1 Smc3 Smc3 Psm3 Smc-3 Smc3/Cap Smc3 Rad21 Mcd1/Scc1 Rad21 Coh- Rad21 Rad21 2/Scc-1 Stromalin Scc3 Psc3 Scc-3 Stromalin/SA Stag1, Stag2 Adherin Scc2 Scc2 Mis4 Pqn-85 Nipped-B delangin/NIPBL Scc4 Scc4 Ssl3 Mau-2 Mau-2 Mau-2/Scc4 Pds5 Pds5 Pds5 Evl- Pds5 Pds5A, Pds5B 14/Pds- 5 Eco1 Ctf7/Eco1 Eso1 Eco/Deco Esco1, Esco2

Table 3: Adherin and cohesion subunits are conserved. Names of genes as reported for these five species are shown (Adapted from Dorsett et al 2006).[2]

Scc2 = NIPBL homolog

Figure 3: A model of how Scc2/Adherin facilitates chromosomal binding of cohesin (S. cerevisiae.) Binding of the origin recognition complex proteins, Mcm 2-7 and Cdt1 are proposed to recruit the binding of adherin to DNA. Adherin stimulates the opening of the cohesin ring for entry of the chromosome. Cohesin binds at adherin binding sites and can be moved by transcription. Prophase removal of adherin was shown to be done by Cdc2 and not by Polo-like kinase aurora B. The possibility also exists that cohesin itself may influence transcriptional status and act as a transcriptional boundary. (Adapted from, Dorsett, D., 2004.) 14

The exact role of NIPBL in sister chromatid cohesion is not known but there have

been models to propose its function in loading cohesion. Mutations in S. cerevisiae Scc2

and S. pombe Mis 4 genes produce precocious sister chromatid separation.[24]. Scc2 is

purported to aid in the loading of cohesion onto DNA (figure 3) [32, 33]. NIPBL was

initially linked to cohesin by previous studies of its homologs. Scc2 and Mis4 studies showed that each were essential for the proper function of cohesin. Scc2 was essential in sister chromatid cohesion while a mutation on Mis 4 resulted in chromosome

missegregation [34]. Subsequent studies in frog eggs, (Xenopus laevis) showed that its

NIPBL homolog XScc2, was essential in the proper functioning of cohesin. The

disruption of the XScc2 binding to cohesin and DNA led to disruption of sister chromatid

binding and disruption of the cell cycle. In Xenopus laevis XScc2 studies, the attachment

of XScc2 onto DNA coincided with the assembly of the replication machinery. Cohesin

has been shown to be concentrated at sites of active transcription. Cohesin was shown to

move from sites of Scc2 loading to sites of convergent transcription [35]. The role of

Xscc2 in recruiting the replication machinery brings to light an additional possible role of

NIPBL in regulating transcription. XScc2 was shown to bind DNA during assembly of

the replication complexes. This in turn catalyzed the loading of cohesin onto the DNA

[24, 36].

Identification of the roles of the NIPBL homologs in chromosome cohesion

hinted at its importance in chromosome cohesion [29]. Chromosomes from mutation

positive individuals (NIPBL mutation) when compared with mutation negative control

individuals showed a significant percent differences in the number of separated sister 15

chromatids. 41% of the mutation positive chromosomes displayed precocious sister

chromatid separation, compared to 9% of the wildtype controls [29].

In separate experiments, Drosophila Nipped-B was found to activate promoters located several base pairs away (85kb) via remote enhancers. This experiment was done initially to identify genes that regulate the cut gene [23]. Nipped-B was shown to participate in the activation of the cut and ultrabithorax(ubx) genes in Drosophila. The cut gene is a known regulator of wing and limb development. The ultrabithorax gene is a homeobox gene activated when cut is hindered by a gypsy insulator with a mutant

Nipped-B. Mutations in Nipped-B led to decreased expression of the cut gene[23].

Regulating a distant gene required interactions between enhancers and promoters and it has been speculated that NIPBL may work in the same fashion. Reduction of Nipped-B through knockdown experiments was shown to reduce activation of the genes. On the other hand, partial reduction of cohesion subunits increased cut activation[37]. This shows that cohesion and Nipped-B have conflicting roles in gene activation. The model

(figure 4) below was designed to illustrate the conflicting roles that cohesion and Nipped-

B have on gene regulation. It is thought that cohesion blocks enhancer-promoter interaction blocking gene activation, while Nipped-B facilitates activation by removing cohesin.[37]

Figure 4: A model of how NIPBL loads cohesin onto chromosomes. It is proposed that NIPBL opens the cohesin ring to load cohesin on chromosomes and also to take it off. The loading or unloading of cohesin by NIPBL determines if a gene can be transcribed. In the case of a mutant NIPBL, the loading or unloading ability of the NIPBL protein would be reduced hence reducing how many times a distant enhancer-promoter function can carry out transcriptional activation. Putting cohesin on is needed for sister chromatid cohesion, and removing cohesin allows enhancer promoter interaction allowing gene activation (Adapted from Rollins, R.A. et al 2004))

Chromatin

Heterozygous mutations of NIPBL cause CdLS. Therefore the varied symptoms displayed in CdLS likely as a result of disrupted gene expression. Chromatin is constantly being remodeled in order to access the tightly wound DNA for gene expression. The packaging of DNA onto chromatin allows the DNA to fit into the nucleus, and more importantly because of its association with Heterochromatim protein 1 (HP1) as described below. The basic unit of chromatin is a nucleosome. During DNA packaging,

DNA is wound onto a histone core of proteins that consists of two copies each of, H2A,

H2B, H3, and H4. The histone core of proteins are assembled into an octamer. Each histone octamer is called a nucleosome. The nucleosome is further compacted into a higher order structure by the linker histone H1.

In condensed chromatin, the accessibility of transcription factors that regulate transcription of genes is controlled. This involves the active unwinding of DNA to expose a certain length of the DNA so that transcription elements could associate and start the transcription process [38, 39]. There are two types of chromatin, heterochromatin and euchromatin. First differentiated in 1928 by a botanist Emil Heitz, heterochromatin were discovered as regions that remained darker stained even after moss cells had passed metaphase while euchromatin stained lighter after metaphase [40]. Aside from being regions of dark staining, heterochromatin also differs from euchromatin in generally being a region of inactive transcription while euchromatin is generally a site of active transcription. However, is now known that some genes that are vital for cell function are located in heterochromatin [41, 42]. When euchromatic and heterochromatic genes are rearranged, gene expression is affected.[43] When euchromatic regions were brought in 18

proximity to heterochromatic genes, those genes in euchromatic regions were observed to be suppressed, a mechanism termed position effect variegation (PEV) [44]. It is now emerging that there are posttranslational histone modifications that have brought to light the roles of chromatin structure and packaging.

Chromatin is important for gene regulation

Histone proteins that aid in packaging of DNA have tails that can be post- translationally modified. Amino terminal tails of histone proteins were found to be chemically modified after they had been synthesized[45]. The type, number and position of the modification of histone tails represented different functional states. The transcriptionally active environments of chromatin were found to be abundant with acetylated histones. Acetylation has been implicated in gene activation and gene silencing depending on which amino acid was acetylated on the histone tail. [46]. In addition to acetylation, histones are also modified by phosphorylation, methylation and ubiquitination. Histone phosphorylation is also known to participate in gene expression.

Histone H3 was observed to be phosphorylated upon stimulation of quiescent cells by growth factors [47, 48]. Ring finger proteins Ring1B and Mel18 are recruited in early development and this is linked to ubiquitinatin of H2A [49]. Histone methylation has also been been linked to transcriptional silencing.[50]

The discovery of a protein HP1 perhaps bridged the gap between gene silencing and chromatin modification. Previous studies that looked into acetylation of histones H3 and H4 found that they appeared to be less acetylated in regions of heterochromatin [51,

52]. Human Suv 39H1, a histone methyltransferase was found to methylate histone 3 at lysine 9 [50]. This phenomenon was conserved among mammalian homologues of 19

Drosophila Su(var)3-9 and of Schizosaccharomyces pombe clr4. The methylation of

lysine 9 of histone 3 created a binding site for binding of a heterochromatin protein,

HP1.The binding of HP1 recruited the binding of SUV39H1 such that there was spreading

methylation and recruitment of HP1 (fig.5.)[53]. Similar experiments supported the role

of HP1 binding to methylated histone 3 on lysine 9. Additional experiments showed that

HP1 had a high binding affinity to trimethylated lysine 9 on histone 3 [54].

Figure 5: A model of heterochromatic self-maintenance by the SUVAR39H1/HP1 complex.HP1 is known to associate with the enzyme SUV39H1. This enzyme recruits the binding of HP1 to histones by selective methylation. This creates heterochromatically repressed regions (Adapted from Bannister, A.J et al. 2001)

The specificity of HP1 binding to methylation sites was illustrated by the

mutation of the chromodomain of HP1 and in the mutation of the histone

methyltransferase (Suv 39H1 HMTase) that methylates histone 3. As a result HP1 binding

to methylated H3 lysine 9 was inhibited[54].

Heterochromatin Protein 1 (HP1)

Heterochromatin protein 1 was initially found in a screen to identify proteins in

Drosophila polytene chromosomes[55]. The salivary gland cells in the larval stages of

Drosophila contain large, multistranded polytene chromosomes. Polytene chromosomes are produced by repeated replication during synapsis without separation into daughter nuclei. Using a bank of monoclonal antibodies raised against proteins in the chromocenter of the chromosomes, HP1 was identified [55]. HP1 was found widely distributed in heterochromatin [55, 56]. There are three HP1 isoforms encoded by different genes but they share domain sequence homology [57-59]. The 3 isoforms of

HP1 are HP1α, HP1β, and HP1γ. The HP1 molecule consists of two domains, the chromodomain and chromoshadow domain. In a study to identify the sequence of

Drosophila polytene chromosome(Pc) proteins, it was discovered that the DNA sequence of the N-terminal end of the Pc gene was similar to the N-terminal end of HP1. The Pc groups of proteins were known to be regulators of homeotic genes. Homeotic genes are important in fly development in that they are responsible for maintaining the anterior- posterior axis and segment identity in metazoan animals. Now that they shared sequence homology with HP1 and both contained a nuclear localization signal, it suggested the direct involvement of HP1 in gene regulation[60]. With the identification of the chromodomain, the chromoshadow domain later followed. The chromoshadow domain 22 was shown to be attached to the chromodomain by a hinge region. The chromoshadow domain nor the chromo domain could not bind DNA individually, but existed as a pair that was found conserved across species [61]. In other words, a whole HP1 molecule had to be intact to function [62]. Despite HP1 implication in gene silencing, the exact mechanism remains unknown.

HP1 binds other proteins by its chromoshadow domain.

With the discovery that HP1 consisted of two domains, recent research by

Lechner and colleagues managed to uncover an interaction between NIPBL and HPI family proteins [63]. They used the fact that HP1 and the interacting proteins all shared a

PxVxL peptide sequence motif. Previous experiments had shown HP1 binding to CAF-1 with the PxVxL motif[64, 65]. HP1 was shown to bind to sp100, LBR, NIPBL, ATRX with varying affinities. All of these proteins were mapped and were shown to share the

PxVxL canonical or variant motif (Table 4). This study revealed an interaction of HP1 and NIPBL. Table 4 below shows several HP1 chromoshadow domain binding partners and their function. Preliminary research by Lechner and colleagues showed that a protein

KAP-1 that had previously been shown to bind to the HP1 chromoshadow (CSD) domain was mutated and titrated along with its wild type against other proteins that shared the binding motif in a dose dependent manner. This competitive binding assay was to show whether the HP1 binding proteins could bind to the HP1 chromoshadow domain at the same time or competed against each other for the same site. It was observed that there was an increased amount of the competing proteins against the wild type KAP-1 and not in the wild mutant KAP-1[12]. This shows that the proteins all bind to HP1 exclusively by the PxVxL motif that they all share. The researchers went further and constructed 23

Bicistronic plasmids that were used to co-express HP1α and three fragments of NIPBL.

Binding of HP1 to NIPBL was shown to be specific as different HP1 CSD polypeptides were still seen to bind to NIPBL. In a subsequent experiment, a point mutant (denoted

VE) was introduced in the PxVxL motif of HP1. It was also shown that HP1 binding to

NIPBL is very specific as there was no binding of protein from point mutant VE[12] .

HP1 Chromoshadow domain Binding Function partner NIPBL Promoter enhancer communication KAP-1 Transcriptional repression CAF-1 Chromatin assembly Sp100 Nuclear body formation ATRX, BRG1 Nucleosome remodeling LBR Nuclear Lamina organization

Table 4: Proteins shown to interact with the HP1 chromoshadow domain. All these proteins have the PxVxL binding motif and have been shown to bind the HP1 chromoshadow domain. Adapted from Lechner et al 2005.

KAP-1 Sp100 CAF-1 HP1 LBR BRG1

Nucleosome CD CSD NIPBL

Basic building block Heterochromatin formation Promoter Enhancer of chromatin Gene silencing Communication/Sister DNA + histones Chromosomal integrity Chromatid Cohesion

Figure 6: An illustration of the HP1 protein and its complex with NIPBL. It shows the two binding domains of HP1, the Chromodomain (CD) attached to the nucleosome and the chromoshadow domain (CSD) to NIPBL and other proteins (KAP-1, Sp100, CAF-1, LBR, BRG1). Refer to table 4 for the function of the proteins.

Lechner and colleagues whose data is shown on figure 7 showed that HP1

physically interacts with NIPBL. The antibody protein complex is analyzed to see if other proteins are also in the complex. On figure 7A, the coomasie blue-stained gel reveals several proteins that precipitate with FLAG-HP1α. Figure 7B illustrated a second coimmunoprecipitation and silver stained gel which is more sensitive. The arrows indicate an HP1γ complex not yet characterized. Proteins on figure 7A were cut out and

mass spectroscopy was used to identify the proteins. To support the identity of the

proteins, a western blot was run (figure 7C.).

ABFLAG-HP1a FLAG-HP1 C FLAG-HP1a wt WA m ag wt VM IK WA NIPBL NIPBL Mi2b (kDa) NIPBL BRG1 BRG1 200 KAP1 CAF1 p150 116 KAP-1 KAP-1 97 CAF1

CAF1 p60 66 flagHP1a

CAF1 p48 46

FLAG-HP1a 30 FLAG-HP1a FLAG-HP1g 21 14 1 2 3 1 2 3 4

Figure 7A: Immunopurified FLAG-HP1α complexes from HEK293 nuclei. The immunoprecipitate wild type flagHP1a complex contains several large polypeptides that are decreased in the CSD point mutant (WA). Polypeptides were identified by mass spectrometry. Coomassie blue-stained gel is shown. B. Similar immunopurification of flag-HP1α and flag-HP1 γcomplexes as in A except that a silver stained gel is shown and the NIPBL polypeptide is highlighted. C. Western blot analyses of flag-HP1α complexes similar to panel A, but also including CD point mutant VM and CSD point mutant (IK). The NIPBL and other proteins are retained with an intact CSD. The point mutant VM had no effect because a mutation at the chromodomain does not affect the binding of proteins to the chromoshadow domain. The arrows indicate not yet identified protein bands. Adapted from Lechner et al 2005.[63]

Heterochromatin protein 1 has been shown to be involved in epigenetic silencing of genes, gene regulation and chromatin remodeling.

Epigenetics is gene regulation independent of the underlying DNA and is

heritable. Chromatin-remodeling factors, histone-modifying enzymes, gene-specific and general transcription factors, and RNA polymerase affect the activation of a gene [66]. X-

chromosome inactivation in somatic cells is stable and the mechanism is proof of

epigenetics. This process consists of the transcriptional silencing, through

heterochromatinization, of one of the two X chromosomes in female mammals, thus

ensuring dosage compensation for X-linked gene products between XX females and XY

males. [67] It has also been shown in plants that chromatin remodeling factors control the

“on and off” states regulating what gene is expressed at what time and occurs with each

stage of development [68]. In Drosophila, it has been shown that the Polycomb group of

proteins (PcG) are transcriptional repressors of homeotic genes while Trithorax group of

proteins (trxG) are transcriptional activators and that the two groups of genes maintain

what is called a “locked” state in which the genes can be expressed or not expressed.

[69].

When histone tails are modified, they are said to be epigenetically marked by the

modification be it methylation or acetylation. This modification has been shown to affect

the expression of genes by maintaining a chromatin state that is heritable. The gene that

coded for the HP1 protein had earlier been identified as being coded by Suvar(205) a

gene that was involved in suppression of PEV. A mutation of Drosophila Suvar(205), a

gene that coded for HP1, there was disrupted splicing of the HP1 mRNA showing that

HP1 heterochromatin protein played a role in PEV. [70] 29

The discovery that the modification of histone tails were epigenetic markers,

brought a new way of understanding and thinking into the role chromatin structure played

in regulating gene function. There was no one way in which genes were regulated.

Instead, the collective modification of histone tails brought about patterns later described

as the histone code hypothesis [71]. The histone code was based on the fact that the type number and location of histones existed a state of equilibrium, such that should this equilibrium be disrupted there would be changes in the modification of histones. In yeast, the histone acetylatransferase GCN5 is an activator of PHO5 while the histone deacetylase RPD3 is a repressor of PHO5. By measuring the acetylation state around the

PHO5 gene, depletion of either RPD3 and GCN5 led to changes in acetylation or

deacetylation of the cell. [72]. On the other hand, removal of the destabilization restored

the initial state of equilibrium (acetylation and deacetylation.).

Malfunction/mutation of chromatin proteins is the basis for a number of disease syndromes.

Roberts syndrome has been reported to be similar to CdLS[73]. Symptoms of

Roberts syndrome such as growth retardation, microcephaly, deformed facial features,

and malformation of the arms are shared with CdLS. Whereas CdLS is associated with a

mutation of the NIPBL protein, a mutation of ESCO2, a cohesin loading factor, (see table

3) is associated with Roberts syndrome [73]. It has been observed that in invasive

metastatic breast cancer cells, HP1 is down-regulated compared with poorly invasive

non-metastatic breast cancer cells. This down regulation is also facilitated by lack of

dimerization of the HP1 rather than a mutant chromoshadow domain [74, 75]. Chromatin

proteins are relied upon to coordinate gene expression and other genomic events. Based

on HP1 and its physical interaction with NIPBL, and the knowledge that mutation of 30

NIPBL leads to CdLS, a multisystem disorder, it is clear that understanding the nature of

their interaction would lead to a better understanding of the epigenetic and chromatin-

based regulation of genomic processes that contribute to development.

Rationale, Hypothesis and Experimental Objective.

CdLS is a complex disorder in which multiple processes are involved. Multiple organs/tissues are affected, suggesting that multiple cellular processes malfunction. A mutant NIPBL has been shown to cause sister chromatid cohesion defects [29]. This is in line with mutants in NIPBL homologs that the sister chromatid cohesion role is conserved among species (see table 2). A second role of NIPBL is in gene regulation as has been shown by its Nipped-B homolog. On one hand NIPBL is directly associated with chromatin while playing a role in the loading of cohesion, and on the other hand it

has gene regulation and transcription regulation functions. Nipped-B was shown to act as a distant enhancer of a gene 85kbs away. NIPBL protein can be thought to maintain a balance in regulating distant genes as in Drosophila but is all dependent on the protein level. Because developmental symptoms of CdLS are caused by a heterozygous loss of function of NIPBL due to mutation, it likely reflects gene expression changes as opposed to cohesin or repair defects. Since CdLS is characterized by varied symptoms, NIPBL is suggested to regulate multiple processes or the same process in different tissues.

Coordinating gene expression and other genomic events relies on chromatin proteins. As we have seen earlier, epigenetic and chromatin-based regulation contribute to control of development. One of those proteins found to actively participate in both

epigenetic and chromatin-based regulation is HP1. HP1 has been shown to bind NIPBL

[12]. Therefore if NIPBL is to regulate multiple processes, its binding to HP1 provides a 31 link to the nucleosome such that it can affect the transcription of multiple genes. One hypothesis therefore is that NIPBL and HP1 form a complex that regulates gene expression and that this complex is disrupted in CdLS. This could explain why a mutant NIPBL could cause CdLS. To study the role of NIPBL, it is important to first characterize the protein. This can be done by immunostaining and observing the morphological characteristics of cells and also checking the expression levels of the

NIPBL protein.

Materials and Methods

Plasmids: pEGFP-NIPBL was constructed from subsequent restriction enzyme digestion followed by ligation. Courtesy of Dr. Ian D. Krantz and Matthew Deardorff, we obtained the complete NIPBLa cDNA plasmid which was about 8.6kb and had been cloned into a pBSII (Bluescript) which has a bacterial promoter. Both pBSII-NIPBLa and pEGFP-C3 vector (GenBank Accession #:U57607) were digested with BamH1 and Kpn1. A Nipbla fragment of 8.6kb was ligated with the linear pEGFP-C3 using the standard ligation protocol provided by the company that provided the ligase(T4 DNA ligase, Invitrogen,

Calabasas, CA).

Cell culture and transfection: NipblΔ (Cdls 005P, 006P, 185P) and Nipbl wildtype cell lines (controls 1,2, and 3) were grown in RPMI 1640 media (Cellgro/mediatech

Manassas, VA) supplemented with 2 mmol/L L-Glutamine, 100U/ml penicillin-100

µg/ml streptomycin (Pen-Strep), and 10% Fetal Bovine Serum(FBS). Human embryonic kidney cells (HEK293), and Human osteocarcoma cell lines (U2Os) were grown in

DMEM media supplemented with L-Glutamine, Penn-Strep and Calf Serum (CS)

(Cellgro/Mediatech, Mananssas, VA). Cells were passed based on confluence after digestion with 0.05% trypsin. Cells were cultured in a humidified atmosphere with 5%

CO2. Transient transfection of pEGFP-NIPBL into HEK 293 cells was done at 70% confluence using Lipofectamine (Invitrogen, Calabasas, CA) according to the manufacturers’ protocol. 2ug of DNA was mixed with 300ul of lipofectamine in a 6 well plate. The cells were harvested 48hrs later for western blot analysis.

Immunoflourescence Analysis: Cells were grown on tissue culture plates with

coverslips. Coverslips were carefully removed with sterile forceps and placed in a 24 well

plate. The cells were washed twice with 500ul 0.05%Tween 20 in phosphate buffered

saline, PBS(PBS-10X phosphate buffered saline - 800 g NaCl, 20 g KCl, 144 g Na2HPO4 and 24 g KH2PO4 in 8 L of distilled water). The cells were pretreated with 0.1% Triton

X-100 in PBS for 5mins. Preliminary experiments showed that the pretreatment stage

was necessary for optimal permeabilization of the cells. The cells were fixed with 2%

paraformaldehyde in PBS for 5minutes. A second 0.1% Triton-X100 in PBS treatment

was carried out for 10minutes. This was followed two washes with PBS with 0.05%

Tween (PBST). For antigen detection, 250ul of primary antibody diluted to 1:1000 depending on manufacturer or investigator recommendations was incubated with the fixated cells on coverslips for 1hr. PBST was again used twice to wash the cells. To detect the primary antibody via fluorescence, 250ul of a fluorescent-conjugated secondary antibody was incubated with the fixed cells for 30mins. The secondary antibody was either a 1:1000 dilution of Alexa-flour 488 rabbit/mouse or Alexa-flour 594 rabbit/mouse(Invitrogen, Carlsbad CA) in PBST with 1 % BSA. The coverslips were washed once with PBST and mounted with Antifade mounting media (Molecular

Probes/invitrogen, Carlsbad CA) with DAPI as a counterstain for DNA.

Doubling time: Both NipblΔ (Cdls 005P, 006P, 185P) and Nipbl wildtype cell lines

(controls 1,2, and 3) were grown in RPMI 1640 media (Cellgro/mediatech Manassas,

VA) supplemented with supplemented with 2 mmol/L L-Glutamine, 100U/ml penicillin- 34

100 µg/ml streptomycin, and 10% Fetal Bovine Serum(FBS). Cells were subcultured and

growth ratewas measured against time in hrs.

Western blot analysis: Following cell culture, plates were treated with differing RIPA buffer amounts(150mM NaCl, 25mM Tris-HCL pH 8, 0.1mM EDTA, 1.0% Nonidet p40,

0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate) based on plate diameter. For 10cm cell culture plates, following cell culture, they were washed 3 times with 10mls PBS. The washed plates were placed on ice and 500ul ripa buffer which contained a protease inhibitor cocktail (1mM phenylmethylsulphonyl fluoride, 1ug/ml leupeptin, 1ug/ml pepstatin, and 1mM dithiothreitol). The lysates were kept on ice for 10 minutes, vortexed once, and centrifuged at 4°C for 20 minutes at 14,000 x g in an Eppendorf

microcentrifuge. Supernatants were transferred to sterile tubes, and protein concentrations were determined using the Bradford assay. 1X Laemmli buffer (10% glycerol, 2% SDS,

50mM Tris-HCL, pH 6.8, 360mM β-mercaptoethanol, 0.05% bromophenol blue) as

added to the cell extracts and heated at 90-1000C for 15mins. Cell extracts (20 µg-100ug)

were electrophoresed using a 4-12% bis-tris polyacrylamide gel. (Invitrogen.)

Electrophoresis was carried out at 200V for 1hr. After electrophoresis, gels were

transferred for 2 hours at 48mA/m2 onto Polyvinylidene fluoride (PVDF) membrane

(Millipore, Bedford MA). Subsequently, blots were probed by using primary antibodies

and the corresponding secondary antibody. The protein bands were visualized using the

alkaline phosphatase reagents BCIP(5-bromo-4-chloro-3-indolyl phosphate), and

NBT(nitro blue tetrazolium) from Amersham Biosciences (Arlington Heights, IL).

Antibodies: Anti-HP1α, anti-HP1β and anti-NIPBL mouse monoclonal antibodies used as primary antibodies in immunoflourescent staining were derived from the Wistar

Institute, Philadelphia, PA. Anti-mNIPBL monoclonal antibodies 14.5.2 and 5.2.1 that were affinity purified from hybridoma cell line secreted antibody from the Wistar institute, and anti-rNIPBL 3472 and 3473 were used. Anti-NIPBL rXScc2N used on the

Western blot and Immunoflourescence staining was a gift from Dr. Kyoko Yokomori

(UC, Irvine.).

Recombinant proteins: 6his-IDN3 and GST-IDN3 control proteins, as described previously, were derived from the 906-1091aa sequence that was bacterially expressed from the full length NIPBL.[76]

Mouse protein heart extracts: Three 24week old female NOD/SCID mice were sacrificed and their hearts surgically removed. The hearts were fit into 1ml cryotubes and snap frozen in nitrogen. 20mls liquid nitrogen was placed onto a pestle and mortar to precool it. Using forcepts the mouse hearts were taken out of the cryotubes and put onto the pestle with 20mls nitrogen. The heart pieces were ground into a powder with the mortar. It was poured into a 15ml tube and put on ice immediately. After 5mins, 1ml

RIPA buffer (150mM NaCl, 25mM Tris-HCL pH 8, 0.1mM EDTA, 1.0% Nonidet p40,

0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate) with protease inhibitors was added into the 15ml tubes. It was sat on ice for 30mins with occasional mixing every 6mins. It was vortexed and transfer to a 1.5ml tube to be centrifuge at maximum speed (1500rpm,

40C) for 20 minutes. The supernatants were transferred to a new 1ml tube. 20ul of the 36

sample were aliquoted in a separate tube for protein quantification. Western blot analysis

was carried out as described above.

Metaphase spreads: Metaphase spreads of human fibroblasts and mouse embryo were

prepared from cells cultured for 12hrs in media supplemented with 0.1ug/mL of colchicine. Trypsinized cells were pelleted, subject to incubation in 75mM KCl for

20mins. at 37oC, then re-pelleted and fixed in 3:1 methanol/acetic acid. Metaphase

spreads were obtained by dropping cells resuspended in 3:1 methanol/acetic acid onto

glass slides and staining with DAPI or Giemsa. Sister chromatid cohesion level was

scored in three categories if the majority of sister chromatids (>50%) in the metaphase

spread have the following characteristics: separated - completely separated and no

connection at the centromere; loosened - separated but with the connection remains at the

centromere and closed if the is no gap and chromatids remain connected. 100 metaphases

per glass slide were counted. Three individuals scored the same slides fibroblasts and

scoring was blinded with respect to Nipbl status.

Results

Specificity and colocalization of anti-NIPBL and anti-HP1 antibodies by immunostaining.

Mutation of NIPBL has been associated with CdLS. CdLS symptoms are likely to be reflected by changes in cellular characteristics internally. It has been shown that cells

from patients with CdLS display precocious sister chromatid separation. It has previously been reported that HP1 and NIPBL coimmunoprecipitate.[12] To establish the colocalization of NIPBL and HP1, U2Os cells were grown on coverslips then stained by indirect immunoflourescence using specific antibodies to endogenous HP1 and NIPBL

(figure 8). On the first and second row, the same cells were stained with mouse HP1 α and β and rabbit (red) XScc2N. As expected we saw colocalization (yellow). We then used the anti-NIPBL mouse antibodies shown in green in row 3 of figure 8. Areas of

colocalization (yellow) are observed when the anti-NIPBL overlapped with the HP1

antibodies. Both the mNIPBL and rXScc2N had a similar colocalization with the HP1

antibodies in the nucleus.

Characterization and morphological characteristics of CdLS cells.

To demonstrate the morphological characteristics as a result of cells having a

mutant NIPBL, we sought to use physical observation of growing and immunostaining of

the cells. Through our collaborators at the Children’s Hospital of Pennsylvania (Dr. Ian

D. Krantz and colleagues) we obtained human fibroblast cells from patients with CdLS

and a NIPBL mutation and control cell lines from patients without CdLS and no NIPBL

mutation. The cell lines are CdLS-185P, CdLS-005P, CdLS-006P and control cell lines

CON1, CON2, CON3 (figure 10). Phase contrast images were taken of subconfluent 38 cultures. Growth rates of both the mutant and wild type were determined by taking cell counts each day and calculating the doubling time. Labeling cells with a fluorescent

NIPBL antibody to identify its phenotypic characteristics would provide a firsthand way to track changes in cell constituents and localization of the NIPBL proteins. It was determined that NIPBL localized in the nucleus along with HP1. The NIPBL mutant cells exhibited a ‘wrinkling’ phenomenon less observed in wild type cells and grew slower.

Figure 8: Colocalization of NIPBL and HP1 in U2OS cells. Detection of NIPBL and HP1 in U20S cells. NIPBL, HP1α and HP1γ signals (green) derive from mouse monoclonal antibodies. Dual staining with rabbit antibodies to NIPBL (αSCC2N) are shown as red signals. The overlap shows colocalization of NIPBL and HP1 proteins. Localization is predominantly nuclear with negative nucleoli. The 10Ab were diluted 1:1000 while the secondary 20Ab was diluted 1:10,000.

Characterization of the morphological characteristics of human fibroblast cells with mutant and wild type NIPBL.

To determine the extra-cellular characteristics of mutant versus wild type cell

lines, NIPBL mutant cell lines CDLS-185P, CDLS-005P, CDLS-006P and wild type cell

lines control 1 and control 2 were grown in similar conditions on plates (Table 5). The cells were visually inspected via phase contrast images recorded with a digital camera.

There were no observable differences between the mutant and wild type cell types. It was

realized that the mutant cell lines in general took a longer time to grow. To quantify this,

the doubling time of the cell types were quantified in hours. Since mutant cell types

exhibited differing morphological characteristics from the wild type cell lines, to

determine if it was as a result the nature of the NIPBL protein being mutant or not,

NIPBL antibodies were used to stain the cells. The mNIPBL antibody showed a more

granulated nuclei mostly in the NIPBL mutant cells. Rabbit anti-Scc2N antisera staining

of the cells exhibited either a ‘smooth’ or ‘wrinkled’ nuclei(Table 5 and figure 9).

Figure 9 illustrates a larger nuclei sampling of the CDLS cells stained with the rabbit

anti-Scc2N antisera. To show the extent of nuclei wrinkling, a larger sample of mutant

and wild type CDLS cells were observed (figure 9). Nuclei bearing the ‘wrinkles’ were

counted and the results summarized in Table 6. On average, 63% of the mutant cell lines

exhibited the ‘wrinkling’ phenotype versus 27% of the control cell lines which was a

statistically significant difference.

(hrs)

Table 5: Morphological differences between mutant and wild type human fibroblast CdLS cells. Phase contrast cells taken with a camera from a light microscope. Growth rate measured as 1 doubling against time in hrs. ‘Wrinkled” nuclei observed as a result of the SCC2N staining antibody were counted as a percentage of the total. Scc2N is human NIPBL antibody raised in rabbits. Mouse αNIPBL showing nuclear localization. Observed more prominent antibody staining in control cells as compared to CdLS mutant cells.

CdLS-185P

CdLS-005P

CdLS-006P

CONTROL 1

CONTROL 2

CONTROL 3

Figure 9: Immunofluorescent staining of CdLS and control human fibroblast cell lines. Fluorescent micrographs show the staining pattern revealed by the SCC2N anti-NIPBL antibody, which is made evident by a ‘wrinkling’ phenotype in some nuclei. Nuclei bearing the ‘wrinkles’ were counted and the results summarized in the Table 6 below. Statistical tests, by 2-way ANOVA with replication, of the above data if all mutants and controls are grouped together show a p-value of 0.6>α (0.05).

Cells wrinkles smooth total % wrinkled CDLS-185P 35 38 73 48 CDLS-005P 52 8 60 87 CDLS-006P 72 65 137 53 CONTROL 1 33 65 98 34 CONTROL 2 35 126 161 22 CONTROL 3 15 45 60 25 *Average wrinkled is 63% from CDLS cell lines and 27% from control cell lines.

Table 6: Characterization of NIPBL nuclear staining morphology with rabbit anti-Scc2N antisera.

Characterization of anti-NIPBL antibodies in cell lines by Western blot analysis.

NIPBL is a chromatin protein associated with the congenital disorder CdLS.

Having shown by immunoflourescent microscopy the localization of the NIPBL antigen, we set out to examine the NIPBL protein by Western blot analysis. Protein expression levels were detected using affinity-purified primary antibodies against NIPBL (figures

10A, 10B and 11). In figures 10A and 10B two identical SDS page (4-12% bis-tris,

Invitrogen) gels were run. As positive controls, purified NIPBL recombinant proteins

GST-IDN3 and 6-HIS-IDN3 were included in the western blot[12]. In addition, separate extracts from cells transiently transfected expression plasmids pEGFP-C3 and pEGFP-

C3-NIPBL was also included. We hypothesized that the recombinant plasmid pEGFP-

C3-NIPBL would show increased NIPBL levels upon completion of western blotting.

Contrary to our expectations, only the positive control lanes showed detection of protein by the antibodies. Even though the rNIPBL antibody had a greater cross-reactivity with other proteins than the mNIPBL, still there was a detectable specific interaction between the rabbit antibody and the purified recombinant proteins (figure 10B). The rabbit anti-

Scc2N antisera reacts to the GST-IDN3 and 6his-IDN3 proteins because it recognizes its epitope on the ~200aa NIPBL fragment. The specificity of the antibodies to the positive control were summarized on table 7.

Our inability to detect endogenous full length NIPBL which is estimated to be about 300kDa led us to believe that the NIPBL protein was deteriorated or lost by the standard protein extraction and blotting techniques. This led us to use different reagents and techniques to try to detect a protein about 300kd (data not shown). It was possible that we did not have sufficient NIPBL protein on our blots and this was below the 45

detection limit. The standard western blots were run with total protein between 25-50ug.

The blot on figure 12 was a result of cell extract from sacrificed mice hearts. Preliminary data has shown increased levels of NIPBL in the heart tissue. We hypothesized that if we used more of the mouse heart protein in a high concentration of about 100ug, we could detect the endogenous NIPBL protein. A band of about 300kDa was observed on a

Western blot gel with the rabbit polyclonal antibody to rabbit anti-Scc2N antisera (figure

11). This was the first time we had seen a distinct 300kD band. Also on figure 11 we ran a positive control with purified NIPBL recombinant protein 6-his IDN3. There was a strong reaction with a protein of 28kD size which corresponds to the position the 6-his

IDN3 recombinant protein size. Currently this protein is awaiting identification using mass spectroscopy techniques.

Figure 10: Western blots showing the expression of the NIPBL recombinant protein in cell extracts. Western blots were performed on 4-12% bis-tris gels.(invitrogen, Carlsbad, CA.) The blots on panel A were probed with monoclonal mouse anti-NIPBL antibodies 14.5.2 and 5.2.1. The blots on panel B were probed with rabbit polyclonal anti-NIPBL antibodies 3472 and 3473. The antibodies were used in concentrations of 1:500 and 1:5000. Lane 1 is seeblue plus 2 ladder. Lanes 2 and 3 are NIPBL recombinant proteins GST IDN3 and 6-HIS-IDN3 respectively. Lane 4 is a protein extract from the human embryonic kidney cell line 293 transfected with pEGFP-C3 vector while lane 5 was transfected with the recombinant plasmid pEGFP-C3-NIPBL as a control. .

Figure 11: Western blot of mouse heart extracts showing the specificities of two Nipbl antibodies, affinity purified mNIPBL Ab 5.2.1 and rNIPBL Ab XSCC2N. Lane 1 shows the migration of the molecular weight standard seeblue plus 2. Lane 2 has 100ug mouse 2 total heart protein extract, lane 3 has 100ug mouse 1 total heart extract, lane 4 has a positive control recombinant protein 6-his-IDN3 while lane 5 is a negative control that contains the loading dye alone.(Repeated and sent for mass spectroscopy)

Names Antibody Dilution Antigen Types(species) 56kDa 28kDa and types. GST- 6HIS- locations IDN3 IDN3 binding binding GST- mNIPBL 1:500 NIPBL MoAba Specific Specific NIPBL 14.5.2 protein (a.a. 906- mNIPBL 1:5000 NIPBL MoAba Specific Specific 1091) 14.5.2 protein mNIPBL 1:500 NIPBL MoAba Specific Specific 5.2.1 protein mNIPBL 1:5000 NIPBL MoAba Specific Specific 5.2.1 protein

GST- rNIPBL 1:500 NIPBL PoAbb Specific Specific NIPBL 3473 protein (a.a. 906- rNIPBL 1:5000 NIPBL PoAbb Specific Specific 1091) 3473 protein rNIPBL 1:500 NIPBL PoAbb Specific Specific 3472 protein rNIPBL 1:5000 NIPBL PoAbb Specific Specific 3472 protein 1:1000 PoAbb * Specific rXScc2N NIPBL protein aMouse; *Unclear brabbit

Table 7: Table showing the antibodies used to detect the NIPBL protein. Antibodies mNIPBL 14.5.2, mNIPBL 5.2.1, rNIPBL 3473, rNIPBL 3472 were able to detect recombinant proteins 6-HIS IDN3 and GST IDN3 but not the native NIPBL protein in cells. Antibody rXScc2N was able to detect recombinant protein 6-his IDN3 as well as a native protein presumed to be NIPBL about the 300kD mark pending further analysis.

Analysis of precocious sister chromatid separation in Human and mouse embryo fibroblasts.

To examine the role NIPBL in the stable propagation of genetic material during cell division we set to examine mutant and wild type human and mouse fibroblast cell lines during the metaphase stage. It is at this stage that sister chromatids are highly condensed and still attached. Cells that were grown for 12hrs in media supplemented with

0.1ug/mL colchicine were dropped on glass slides and stained with DAPI (Colchicine arrests cells in the metaphase stage). Misaligned chromosomes were counted and tabulated as shown in figures 12 and 13. To remove bias, three individuals tallied the chromosomes independently and then the data was pooled and graphed as shown. The criterion for scoring the chromosomes was based on the phenotypes illustrated on figure

14. There were significant differences between the wild type and mutant human fibroblast cell lines. Unexpectedly, the mouse embryo fibroblasts did not follow suit.

There were no observable differences between the mutant and wild type mouse embryo fibroblasts (figure 13).

Figure 12: Figure illustrating chromosome misalignment in ∆NIPBL and wild type human fibroblast cells. P6 and N304 are ΔNIPBL mutant cell lines while the control cell lines are the wild type cell lines. Data in the figure are the mean values ± the standard error of sister chromatid cohesion observed from ≥ 100 metaphase spreads prepared from MEFs(mouse embryo fibroblasts). Controls 1 and 2 yield p-value 0.1794 (θ>0.05), indicates no significant difference between the two controls. The tighly bound verses the separated chromosomes was more important in the comparison between the mutant and wildtype cell lines. *Tight chromosomes from the controls are significantly different from the loosened and separated chromosomes. (p<0.05). This could be a result of reduced NIPBL protein in the mutant cell lines.

Figure 13: Figure illustrating chromosome misalignment in ∆NIPBL and wildtype mouse embryo fibroblast cells. Data in the figure are the mean values ± the standard error of sister chromatid cohesion observed from ≥ 100 metaphase spreads prepared from MEFs. No statistically significant differences in category frequency were observed among the wild type and heterozygous mutant Nipbl cells.

closed loosened separated

Figure 14: Figure of an example of determination of chromosome misalignment in cells. Closed conformation correspond to attached sister chromatids. Loosened conformation correspond to slightly detached sister chromatids while the separated conformation corresponds to completely detached sister chromatids.

Discussion

In this thesis, we set to characterize the NIPBL protein that when mutated, was

shown to be associated with the incidence of Cornelia de Lange Syndrome. Very little is

known of the role of the NIPBL protein. Truncation mutations have been correlated with

a more severe phenotype of CdLS[16]. Patients with CdLS display a variety of symptoms. This is proof that it is not enough to know that a gene can cause a particular disease, but it is important to study the expression profile of genes and how they are affected by other proteins. When NIPBL was found to coimmunoprecipitate with heterochromatin protein 1(HP1), we sought to determine the nature of their interaction[63]. This interaction was important because it has the potential to shed light into the link between NIPBL and gene expression. HP1 has previously been shown to interact with chromosomes via a direct interaction with the methylated lysine 9 of histone

H3[50]. Figure 9 illustrates colocalization of HP1 and NIPBL. This provides a molecular insight into what could be the function of NIPBL. Notably, its interaction with

HP1 could result in nuclear reorganization of the chromosome. It could mean that when

NIPBL binds HP1, it leads to a protein conformation change of HP1 that could hypothetically result in either turning on or off of genes depending on whether the transcription proteins can access the nucleosome.

Further evidence of molecular perturbation of the genome arose when NIPBL mutant cells and wild type cells were examined for changes in morphological characteristics. In general the NIPBL mutant cells grew slower than wild type cells as

evidenced by longer doubling times (Table 5). Their nuclei also exhibited a ‘wrinkling’

phenomenon with the rabbit anti-Scc2N antisera. This ‘wrinkling’ phenomenon that is

more prominent in the NIPBL mutant cells in addition to the spotty pattern of staining

54 with the monoclonal mNIPBL antibody could be because of reduced NIPBL protein resulting in reduced chromosomal integrity. The ‘wrinkling’ phenomenon was observed in 63% of the mutant cell lines compared to 27% of the wild type cell lines (figure 9 and

Table 6). These cell based assays likely reflect the clinical observations in patients with

CdLS. Just as the NIPBL mutant cell lines are slow to develop reflects adverse gene expression defects that is analogous to the developmental defects of CdLS. Another way to look at this is to propose that reduced levels of NIPBL lead to lower interaction with

HP1 hence there results gene expression changes.

To examine the expression of the NIPBL protein in cell lines, several αNIPBL antibodies were tested. In figures 10A, 10B and 11, the mouse monoclonal antibodies

14.5.2 and 5.2.1 and the rabbit polyclonal antibodies 3472 and 3473 were raised by Dr.

Lechner against a NIPBL recombinant protein fragment approximately ~200aa long[12].

This same fragment was fused to a GST tag and a polyhistidine tags and produced as purified recombinant proteins of apparent molecular mass 56kDa and 28kDa respectively. The XScc2N antibody used in figure 11 was a rabbit antibody to NIPBL given as a gift by Dr. Kyoko Yokomori (UC. Irvine.). As shown by figures 10A, 10B and

11, all the mouse monoclonal NIPBL antibodies and the rabbit polyclonal NIPBL antibodies cross-reacted specifically to the purified recombinant NIPBL proteins.

However they did not appear to react with the endogenous NIPBL in 293 cell extracts, nor did they react with a protein in the transiently transfected HEK 293 cells with the

NIPBL expression plasmid pEGFP-C3-NIPBL.

Some possible scenarios as to why the NIPBL protein could not be detected are that; (i) the NIPBL protein was chemically modified so the epitope was inaccessible; (ii) the NIPBL protein was degraded such that we could not detect any full length protein,

(iii) there was not enough NIPBL protein to detect a signal; and (iv) the antibodies were not working. To address these issues, we tried several processes that denatured the cell extracts a little bit more such as in trypsin digestion of the protein extract, or adding the cell extracts directly to the protein sample buffers. This was in addition to trouble shooting experiments such as varying blotting membrane pore size during electrophoresis, varying current, SDS gel type or transfer length on a western blot (data not shown). We also tried using less denaturing techniques such as to preserve the NIPBL proteins integrity such as using a sucrose extraction buffer. It seems that the antibodies could not still access the epitopes of the full length NIPBL proteins. This was in contrast to the fact that the same antibodies showed distinct nuclear localization in indirect immunofluorescent images and also were able to detect the NIPBL fragments 6his-IDN3 and GST-IDN3 (figure 10A, 10B and 11). As shown by the indirect immunofluorescent experiments, the intact NIPBL protein could only be detected by immunocytochemistry and not western blots. The antibodies we therefore used were highly selective and the problems we were having was mainly inaccessibility of the epitope when the protein was extracted from cells and processed for Western blotting. Transient transfection of the cells with an EGFP- NIPBL plasmid showed a fluorescent protein that slowly dissipated over time.

Previous research has suggested that in spite of a lot of literature that attempted to explain this phenomenon, the main problem has not been that the epitopes of the proteins have been changed, but because the proteins are inaccessible or not detectable due to degradation [77, 78]. The epitopes therefore may be conditional in that they react at certain conditions and will work well either in its denatured or native state, but not likely both. In our case, it seems that the antibodies were working well in their native state. Our

monoclonal and polyclonal antibodies to NIPBL were generated out of the same ~200kD

fragment generated from almost the middle of the full length NIPBL protein (a.a 906-

1091) [12]. One solution to this problem would be to use a new antibody using a NIPBL

fragment from another part of the full length gene. We obtained a new anti-rabbit NIPBL polyclonal antibody, XScc2N (a gift from Dr. Kyoko Yokomori, U.C. Irvine.) This antibody was directed towards the c-terminal end of the full length NIPBL protein.

Figure 11 is a Western blot using the rabbit anti-Scc2N antisera alongside a monoclonal mouse antibody. Mouse heart extracts were loaded on the gel at 100ug total protein per well. Research into the expression patterns of NIPBL in mouse embryos showed that there was increased expression in the heart [13](MS Lechner unpublished data). This aim of this experiment was to address the issue of reduced NIPBL protein levels in cell extracts and also involved using a different antibody other than the antibodies generated against the ~200kDa fragment. In both cases we are able to see the positive control which was a 28kDa 6-HIS IDN3 fragment. Notably the part of the blot probed with the rabbit anti-Scc2N antisera has a band approximately 300kDa in size.

Since the rabbit antibody was produced from a different fragment from the ~200aa protein used to make the other antibodies, we speculate that it could have accessed the and reacted with the epitope. However, to prove this, the ~300kDa band detected on the gel with the rabbit anti-Scc2N antisera is pending sequencing. It has been sent for mass spectroscopy.

. To further investigate the role of NIPBL in sister chromatid cohesion in human cells, we examined cohesion in human fibroblast cells as well as in mouse embryo fibroblast cell lines (figure 12 and 13). In line with previously published data, NIPBL homologs Drosophila melanogaster Nipped-B, Saccaharomyces cerevisiae Scc2,

Schizosaccharomyces pombe mis 4 and Coprinus cinereus Rad 9 all have roles in sister

chromatid cohesion[79]. Subsequent research into NIPBL was also shown to have a role

in sister chromatid cohesion[29]. The human fibroblast cells showed a significant difference in chromosome cohesion between the wild type and mutant cell lines as expected. However the mouse embryo fibroblast cell lines did not show a significant difference between the mutant and wild type cell lines. This was unexpected because the

NIPBL mutant mouse cell lines were obtained from mice with mutant NIPBL and exhibited characteristic CDLS developmental features in line with their human counterparts(Dr. Arthur Lander, personal communication). The only plausible explanation at this point is that mice are of a different species. There needs to be further research in mouse cell lines. Still, this does not negate the influence of the NIPBL gene in gene transcription. The NIPBL gene has dual roles, one in sister chromatid cohesion and also in gene transcription[80] and possibly other unexplored roles.

Future Experiments.

So far, we have tried to characterize NIPBL and not looked into the nature of its interaction with HP1. It has been shown that HP1 physically interacts with NIPBL[12].

Heterochromatin is now known to be important in chromosome structure and in gene regulation[81]. HP1 proteins are mainly concentrated in heterochromatin and have been characterized as enhancers in position effect variegation and heterochromatic gene silencing and activation[82]. HP1 proteins have also been shown to bind NIPBL by way of its chromoshadow domain[12, 76] A mutant NIPBL is thought to alter gene expression. The way in which NIPBL alters gene expression is unknown. Future experiments will determine if HP1 and NIPBL bind the transcriptional units of the genes that they commonly regulate. However there is no data to show whether a mutant NIPBL

would interact with HP1 the way its wild type complement would. This would be an

important step in understanding the role NIPBL plays in gene expression in human

beings and shed light into why we see the developmental symptoms such as those in

CdLS. Another way is to modulate the mutations in Cornelia de Lange Syndrome by

introducing similar mutations observed in CdLS. Heterozygous mutation of the NIPBL

gene in CdLS is thought to result in reduced NIPBL proteins. Hence we need to modulate

reduced NIPBL by developing tools to reduce endogenous NIPBL.

Recent research has shown that siRNAs to NIPBL effectively knockdown

endogenous NIPBL. Should shRNA constructs not work, we would use the published

siRNA sequences from other experiments. Toyoda et al, use a NIPBL knockdown in cells to study chromatid cohesion. AA knockdown of NIPBL by RNAi were observed to be mitotically delayed.[83] There are now retroviral packaging systems that can be used to effectively deliver the shRNA into cells. This method makes use of the mechanism that viruses use to integrate its genome into its hosts. This ensures shRNA integration into the host genome and since viruses are expected to infect every cell, it is a more effective method for knocking down the target protein[84].

NIPBL is mutated in about 50% of the cases in CdLS. CdLS patients display variable symptoms. Mutated NIPBL hints into disruption of key regulator genes. HP1 proteins play an important role in gene expression. It has been shown as a repressor and activator of gene activity. It has also been shown to interact with several proteins one, of them being NIPBL. It is now emerging that the binding of the proteins to HP1, is thought to alter the gene regulation roles of HP1. To determine how NIPBL alters the gene regulation properties of HP1, we need to identify possible HP1 mediated candidate genes.

Nipped-B studies in Drosophila showed it regulated the cut gene that was 85kb

59 away[79]. We think that NIPBL may not necessarily bind the transcriptional unit to bind the gene but could act as a long range regulatory element. One way to identify candidate genes mediated by both NIPBL and HP1 would be by looking at published materials.

Better yet we could use chromatin immunoprecipitation(CHIP) and microarray plate experiments. CHIP would be advantageous in identifying those regions of DNA bound by NIPBL and HP1 and microarray analysis can be used to identify potential gene candidates mediated by the interaction of HP1 and NIPBL.

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