Gene Expression inCbx1-/- and Cbx1+/+ Mouse Brains and Placentas Lin Wang Degree project inapplied biotechnology, Master ofScience (2years), 2009 Examensarbete itillämpad bioteknik 30 hp tillmasterexamen, 2009 Biology Education Centre and Sub-department ofDevelopment &Genetics, Uppsala University Supervisor: Reinald Fundele 1 List of abbreviation Actβ actin, beta AD Alzheimer's disease Anxa2 annexin A2 Ascl2 achaete‐scute complex homolog 2 B6 C57BL/6 CAST CAST/EiJ Cbx1/3/5 chromobox homolog 1/3/5 CD chromodomain Cdkn1c cyclin‐dependent kinase inhibitor 1C CSD chromoshadow domain EST expressed sequence tag FTLD‐U frontotemporal lobar degeneration with ubiquitinated inclusions H19 H19 fetal liver mRNA H3K9Me2/H3K9Me3 di‐ or trimethylated lysine 9 of histone H3 HMTase histone methyltransferase HP1 heterochromatin protein‐1 IC2 imprinting center 2 Igf2 insulin‐like growth factor 2 IHC Immunohistochemistry Kcnq1ot1 KCNQ1 overlapping transcript 1 LOI loss‐of‐imprinting MGI Mouse Genome Informatics M/M;C/M;M/C;S/M mating between female ×male mice (M stands for C57BL/6; C stands for CAST/EiJ; S stands for M. spretus) MRC Medical Research Council MSP M. spretus Msuit mouse specific ubiquitously imprinted transcript 1 NCBI National Center for Biotechnology Information Nnat Neuronatin Npc2 Niemann Pick type C2 NTF3 neurotrophin 3 Peg3 paternally expressed 3 PEV position‐effect variegation polyQ poly‐glutamine Ptov1 prostate tumor over expressed gene 1 qRT‐PCR quantitative real time PCR Rasgrf1 RAS protein‐specific guanine nucleotide‐releasing factor 1 RFLP restriction fragment length polymorphism SNP single‐nucleotide polymorphism Snrpn small nuclear ribonucleoprotein N Ube2K ubiquitin‐conjugating enzyme E2K9 Usp29 ubiquitin specific peptidase 29 2 Summary Heterochromatin protein‐1 (HP1) is a nonhistone chromosomal protein that enables heterochromatin formation and gene silencing. Additional findings increasingly suggest new functionalities of HP1, such as stabilization of telomeres and control of gene expression, and others. However, the physiological function of HP1 remains unknown. A previous study, in which our laboratory participated, disrupted the murine Cbx1 gene, which encodes the HP1β isotype, and showed perinatal lethality of newborn mice. This is most probably caused by abnormal differentiation of the neuromuscular junctions in the diaphragm, which causes the newborn mice to suffocate. In addition, defective development of the cerebral cortex was observed. In the brain, microarray hybridizations were performed to identify genes that showed altered expression without HP1β; interestingly, a significant proportion of the genes found with differential expression in Cbx1‐/‐ brains are associated with neurodegeneration. Moreover, since HP1 is capable of gene silencing, the former study in placenta focused on loss‐of‐imprinting (LOI) due to homozygous mutation of Cbx1. The genes, Rasgrf1 (RAS protein‐specific guanine nucleotide‐releasing factor 1) and Nnat (neuronatin), showed LOI in a litter mate Cbx1‐/‐ placenta, not in liver and brain. This study was carried out in an attempt to further confirm the previous discoveries. qRT‐PCR (quantitative real time PCR) and immunohistochemistry (IHC) were performed on Cbx1 wild‐type and mutant mouse brains to determine the neurodegeneration‐related genes’ abnormal expression levels. However, rather contradictory results were obtained, except for consistency of two brain genes: Igf2 (insulin‐like growth factor 2) and Anax2 (annexin A2), which were observed to be up‐regulated in Cbx1‐/‐ brains. In the placenta, Rasgrf1 and Nnat were shown to be expressed only in non‐maternal cells by IHC. LOI of Rasgrf1 was tested by RT‐PCR/RFLP (restriction fragment length polymorphism) analyses, but surprisingly, no imprinting of Rasgrf1 was found in the placenta. No RFLP was available for further LOI studies on Nnat. 3 Introduction Heterochromatin protein‐1 (HP1) obtained its name because of its association with heterochromatin (James and Elgin, 1986), the condensed state of chromatin with limited transcription. HP1 was first discovered in Drosophila as a suppressor of the silencing effect of heterochromatin in position‐effect variegation (PEV) (Eissenberg et al., 1990), which is a variegation caused by the silencing of a gene when it is placed near or within heterochromatin. Since this first observation HP1 has been shown to function in heterochromatin formation and gene silencing in many organisms (Fanti and Pimpinelli, 2008). Evolutionary conservation and gene organization Later studies showed that the HP1 family is highly evolutionarily conserved, and that it exists in almost all eukaryotic organisms (Lomberk et al., 2006). The conservation of HP1 function has been shown by cross‐species experiments: HP1 gene in Schizosaccharomyces, which is named swi6, can be successfully replaced by murine HP1 (Wang et al., 2000); besides, human HP1 is able to substitute for Drosophila HP1, rescuing homozygous mutants of Su(var)2‐5, the gene encoding HP1 in Drosophila, from lethality (Norwood et al., 2004). In mammals there are three HP1 protein isotypes, HP1α, HP1β and HP1γ, which are encoded by chromobox homolog 5 (Cbx5), Cbx1, and Cbx3 respectively (Jones et al., 2000). HP1α and HP1β are localized mainly in heterochromatin, while HP1γ is found in both heterochromatin and euchromatin (Minc et al., 2000). Although Cbx1, Cbx3 and Cbx5 encode proteins with such distinct localization patterns, they are approximately 65% identical (Vermaak et al., 2005). Structural features All three HP1 isotypes consist of a conserved N‐terminal chromodomain (CD) and a conserved C‐terminal chromoshadow domain (CSD), connected by a hinge region (Lomberk et al., 2006). The chromodomain binds to di‐ or trimethylated lysine 9 of histone H3 (H3K9Me2, H3K9Me3) (Lachner et al., 2001), the epigenetic marks for gene silencing. The chromoshadow domain can interact with proteins containing the consensus pentapeptide PXVXL (Thiru et al., 2004). The linker is believed to affect the regulation of HP1 protein, such as localization and interactions (Lomberk et al., 2006) (Fig. 1). Although the functions are different, the chromodomain shares identical amino‐acid with the chromoshadow domain (Cowieson et al., 2000). One hypothesis suggests that, based on the fact of high similarity between the two domains, HP1 encoding genes could have arisen from a duplication of one of these domain encoding genes (Lomberk et al., 2006). Fig. 1 The conserved structure of HP1 proteins. Any HP1 isotype is made up of the chromodomain (CD) at the N terminus (N) and the chromoshadow domain (CSD) at the C terminus (C) linked by the hinge region. The functions of different domains are also indicated. 4 Function A wide range of functions have been discovered for HP1. The most common function of HP1 is heterochromatin formation and gene silencing. As mentioned above, this activity involves interaction between the chromodomain and the histone modifications H3K9Me2 and H3K9Me3, the epigenetic marks for gene silencing (Lachner et al., 2001). The chromodomain folds into a globular conformation consisting of an antiparallel three‐stranded β sheet and an α helix in the carboxy‐terminal segment (Ball et al., 1997). The hydrophobic groove formed on one side of the β sheet provides the appropriate environment for the chromodomain to dock onto the methyl K9 histone H3 mark. In fact, it has been proposed that, expect for chromodomain, chromoshadow domain of HP1 also participates in the heterochromatin binding activity. A model has been suggested on this interaction, with an involvement of the specific HP1‐interacting histone methyltransferase (HMTase). In this model, HMTase methylates H3K9, creating a binding siter fo the chromodomain, while interacting with chromoshadow domain. In this way, heterochromatin with a repressed gene activity is formed (Fanti and Pimpinelli, 2008). Apart from heterochromatin formation and gene silencing, HP1 has been identified with several other functions. HP1 mutation in Drosophila leads to multiple fusions of telomeres, causing extensive chromosome breakages, which indicates that HP1 has a function in stabilization of telomeres (Fanti et al., 1998). In mammals, HP1 proteins are found to be involved in recruiting the cohesion complex and kinetochore proteins, the proteins necessary for chromosome segregation at mitosis (Zhang et al., 2007). Studies show that HP1 participates in regulation of euchromatic gene expression, with a correlation with RNA (Piacentini et al., 2003; Brower‐Toland et al., 2007). Besides, one recent study discovered that in mammalian cells HP1β is involved in DNA damage repair together with casein kinase 2 (Ayoub et al., 2008). More and more HP1 interacting molecules have been discovered in recent years; however, the precise functions of the three heterochromatin proteins in mammalian cells are not well known. HP1β, encoded by Cbx1, is the most studied out of these three proteins. Neurodegeneration A previous study from the laboratory has shown that homozygosity for a null Cbx1 mutation causes perinatal lethality of new born mice, which is most likely due to deficient formation of neuromuscular junctions (Aucott et al., 2008). Moreover, the Cbx1‐/‐ fetuses exhibited abnormal cerebral cortex development, associated with reduced proliferation of neuronal precursors, widespread cell death and edema; the in vitro cultures of neuroshperes from Cbx1‐/‐ brains indicate a severe genomic instability (Aucott et al., 2008). In order to identify