The EMBO Journal vol.6 no.8 pp.2393-2399, 1987 Chromatin from transcribed genes contains HMIG17 only downstream from the starting point of transcription Tomislav Dorbic and Burghardt Wittig analyzed the DNA with respect to three active genes (Dorbic and Wittig, 1986). In employing this well-defined and highly specific Institut ftir Molekularbiologie und Biochemie, Freie Universitiit Berlin, Amimallee 22, D-1000 Berlin 33, FRG tool we were able to locate HMG17 in the neighbourhood of the transcription starting point of three chicken genes from the re- Communicated by A.Rich spective tissue expressing them. Monoclonal antibodies specific for the non-histone chromo- somal protein HMG17 were used to isolate oligonucleosomes from the transcriptionally active chromatin of chicken liver Results and oviduct. The distribution of HMG17 with respect to the For our analyses the genes for vitellogenin II (active in liver, coding region of three genes was analyzed in these oligo- inactive in oviduct of laying hens), lysozyme and ovalbumin (both nucleosomes by employing two independent experimental active in oviduct, inactive in liver of laying hens) were chosen approaches. In the vitellogenin II gene (active in liver) and (Grez et al., 1981; Bellard et al., 1982; Anderson et al., 1983; the lysozyme and ovalbumin genes (active in oviduct) HMG17 Geiser et al., 1983). From the chromatin containing these three was found only downstream from the respective starting genes nucleosomal material was subjected to indirect immuno- points of transcription. The transition from HMG17-free to precipitation with HMG17-specific antibodies and solid-phase HMG17-containing chromatin is located at the transcription bound protein A (Immuno-Precipitin). Parameters which are im- start. This directly demonstrates that the distribution of an portant for these kinds of experiments, such as the size distri- abundant nuclear protein correlates with the observation of bution of the immunoprecipitated oligonucleosomes, their moderate DNase I-sensitivity in upstream regions and of high quantitative recovery, and the enrichment of transcribed se- sensitivity in the coding regions of active genes. quences in each size class, have been exactly defined previous- Key words: chromatin/oligonucleosome/vitellogenin gene/lyso- ly. The procedure yields three fractions SUP, HMG-N and PEL syme gene/ovalbumin gene/transcription (Dorbic and Wittig, 1986). The supematant fraction SUP, in prin- ciple, includes all the mono- and oligonucleosomes not contain- ing HMG17. But due to non-saturating antibody concentration (in order to avoid unspecific binding) SUP also contains some Introduction HMG-N material. HMG-N is the material that can be eluted In recent years it has been repeatedly reported that the preferen- by 0.4 M salt from the tetrameric complex of nucleosome: tial sensitivity of active chromatin domains to DNase I cleavage HMG17:antibody:Immuno-Precipitin. The salt incubation dis- coincides with the presence of the non-histone proteins HMG17 rupts the bonds between nucleosome and HMG17 and yields and HMG14 (Weisbrod et al., 1980; McGhee and Felsenfeld, nucleosomal particles which, up to at least nucleosome repeat 1980; Cartwright et al., 1982; Igo-Kemenes et al., 1982). The number N6 or N7, originate entirely from active chromatin. PEL most convincing evidence supporting the significance of this cor- is the fraction remaining after release of HMG-N. Its main con- relation came from experiments which demonstrated loss and stituents are subnucleosomal particles (Varshavsky et al., 1978) regain of the sensitivity concomitantly with the removal and enriched for transcribed nucleotide sequences but also chromatin restoration of HMG17/14 (Weisbrod et al., 1980). Higher resol- rendered insoluble during incubation. ution studies revealed that DNase I-sensitivity can be further dif- From SUP, HMG-N and PEL the DNA was extracted, sep- ferentiated into a region of high sensitivity belonging to the arated by agarose gel electrophoresis, and hybridized with the expressed coding sequences and adjacent moderately sensitive short RNA probes vitD, lysD and ovaD. These probes are com- regions extending several thousand basepairs upstream and be- plementary to sequences downstream from the transcription start- yond the 3' end of the respective genes. It was concluded from ing point of the vitellogenin II, lysozyme and ovalbumin genes, indirect evidence that HMG17/14 may be responsible only for respectively. The results are shown in Figure 1. the high DNAse I-sensitivity and that the moderate sensitivity The bands in the three tracks labelled 'Inactive Chromatin' rather depends on higher order chromatin structure (Stalder et represent hybridization to DNA of mono- and oligonucleosomes al., 1980; Kuo et al., 1980; Weintraub et al., 1981). However, from an oviduct and a liver SUP fraction. The oviduct DNA was the subject is a matter of controversy (see e.g. Nicolas et al., probed for vitellogenin II and the liver DNA for lysozyme and 1983; Seale et al., 1983; Stein and Townsend, 1983; Swerdlow ovalbumin, i.e. where the respective genes are not transcribed. and Varshavsky, 1983), in part due to experimental discrepancies The ladders of these bands reflect the known size distribution a but also caused by an extrapolation of data obtained from well- of DNA fragments from MNase-digested chromatin having defined components, like chicken erythrocyte mononucleosomes, repeat length of 196 bp. As shown by other authors for ovalbumin et al., to more complex systems. Nevertheless, studying nucleosomal (Bellard et al., 1982) and heat shock protein hsp70 (Wu organization within domains of transcribed chromatin requires 1979), we also observe here that in their non-transcribable state understanding of these phenomena. In approaching this goal we the three genes exist within a regularly spaced nucleosomal array. produced HMG17-specific monoclonal antibodies in order to Being present in SUP fractions proves that these genes in this not able isolate HMG17-containing mono- and oligonucleosomes and state do not contain HMG17, and consequently we were 2393 IRL Press Limited, Oxford, England T.Dorbic and B.Wittig Inactive Chromatini Active -I -r Chromnatin Oviduct L^ ilye r1 IJ i v ejI'__I___ t ---I Oviduc Ovid utc t F - v I_ V 1t .1) LjV.SD ov<L), v .ir D ovD I .1 1,. 1- -X- -I SUP SLiP SLIP SUP HMG-N PEL SUP HMG-N PEL SJP.HMG-N PEI., N7- N 6;- N,3 -. 4pol N :i -. .bw ais NiN2 - Iv-r '. ... - N - 14 .!i N1.- Em.m t-1Cl *j Fig. 1. Hybridization of nucleosomal DNA with RNA probes complementary to nucleotide sequences downstream from the respective transcription start of vitellogenin II, lysozyme and ovalbumin genes. Inactive chromatin: the probe, vitD, for vitellogenin II used with chromatin fragments from chicken (laying hen) oviduct, a tissue in which this gene is not transcribed. Likewise, probes lysD and ovaD for lysozyme and ovalbumin genes respectively, used with chromatin fragments from chicken (laying hen) liver, a tissue in which they are not transcribed. MNase digestion: 2 x 10 nuclei/ml; 5.5 U MNase/108 nuclei. 37°C, 25 min liver, 40 min (oviduct). Active chromatin: the vitellogenin II gene probed in chicken (laying hen) liver chromatin, in which it is transcribed, and the lysozyme and ovalbumin genes probed in chicken (laying hen) oviduct, in which they are transcribed. MNase digestion: 2 x 108 nuclei/rnl; 5.5 U MNase/108 nuclei. 37°C, 10 min (liver as well as oviduct); probe vitD; position +58 to + 138 in the chicken vitellogenin II gene; probe lysD: postion + 190 to - +300 in the chicken lysozyme gene; probe ovaD: position + 155 to - +250 in the chicken ovalbumin gene; SUP, supematant after indirect immunoprecipitation of chromatin with HMG17-specific monoclonal antibodies; HMG-N: oligonucleosomes (that previously contained HMG17), dissociated from the immunoprecipitate by 0.4 M NaCl; PEL, pellet remaining after dissociation of HMG-N; NI, N2, N3, etc. refer to nucleosomal repeat units, NC to core particles, <C to subnucleosomal particles. to detect any significant hybridization in the corresponding HMG- Figure 2 depicts hybridization to DNA from oviduct chromatin N tracks (Dorbic and Witting, 1986). fractions. Three different RNA probes are used, complementary The tracks denoted 'Active Chromatin' represent hybridization to sequences upstream from the starting point of transcription to SUP, HMG-N and PEL fractions from liver and oviduct chro- of the ovalbumin gene. Figure 2A shows the autoradiograph of matin, but in these cases the gene for vitellogenin II was probed the hybridization pattern and Figure 2B outlines the rationale of with vitD in liver DNA and the genes for lysozyme and ovalbu- the experiment as well as the interpretation of the data. min probed Analysis with lysD and ovaD in oviduct DNA, i.e. where the of the DNA in HMG-N with probe ovaUI (extending from - respective genes are actively transcribed. A detailed comparison -1320 to - -1000) revealed the complete loss of all bands cor- of the bands in tracks 'Active Chromatin' may be summarized responding to DNA < 1200 bp, i,e. there is no HMG17 present by the following statements. Firstly, although for each gene one closer than 1200 bp from the probe ovaUL. Conversely, only this band is missing in HMG-N which is present in SUP, more fre- DNA is present as bands in SUP which, since it was not bound quently we find bands in HMG-N which are missing in SUP. by an antibody, represents chromatin not containing HMG17. Secondly, in some cases 'corresponding' bands in SUP and However, as indicated in Figure 2B one cannot decide whether HMG-N appear slightly shifted in position. Thirdly, in com- the hybridizing oligonucleotides in HMG-N run or parison with upstream the regular nucleosomal ladder of hybridization downstream of the probe position on the basis of a single set of patterns (see the reference tracks 'Inactive Chromatin') the three data. We therefore hybridized the oviduct HMG-N fraction with HMG-N tracks display pronounced differences in spacing and the probe ovaUll which extends farther towards the transcription the size distribution of bands.