We have previously reported the mobility of positioned nucleosomes on sea urchin 5S rDNA. In this study we demonstrate the temperature dependence and the range of this mobility on 5S rDNA constructs. We find that this dynamic behavior also applies to bulk mononucleosomes and nucleosomes reconstituted onto sequences of the Alu family of ubiquitous repeats. We conclude that short range sliding is potentially a general phenomenon that is dependent on the underlying sequence and its position on the histone octamer. The nucleoprotein gel analysis used also reveals the dramatic effect on gel electrophoretic migration caused by the location of the histone octamer on DNA fragments. The usefulness of this technique for studying nucleosome positioning and its dynamics is demonstrated.
We have previously identified a generally occurring short-range mobility of nucleosome cores on DNA in relatively low ionic strength conditions. Here we report that this mobility of histone octamers positioned on constructs of 5S rDNA is suppressed by the binding of histone Hi or H5 to the nuceosome. Histone HS Is the more potent inhibitor of nudeosome mobility, in accordance with its higher affinity for chromatin. We propose that this reversible restraint on chromatin dynamics may play a role in local regulation of processes that require access to the DNA. H1 has been identified as a general repressor of transcription (6, 7). It was assumed previously that active genes were devoid of H1 and that Hl-induced higher-order structures caused repression. Recent studies, however, show that H1 remains in active chromatin fractions, probably in reduced amounts or with aweakened afflnity (8)(9)(10)(11). This modified association ofHi with active chromatin may allow access to the DNA.It should be noted that even without H1, the packaging of DNA into nucleosome cores in itselfrenders a large component of the DNA sequences inaccessible to trans-acting factors (for reviews, see refs. 12 and 13). The mechanisms of eukaryotic DNA processing probably involve a dynamic behavior of the nucleosome structure. Various modes of nucleosome disruption, nucleosome transfer, and histone dissociation have been invoked in models for initiation and elongation of transcription (for reviews, see refs. 13 and 14). We have identified (iS) a general mobility of nucleosomes under conditions of relatively low ionic strength that may be relevant to these models. Nucleosome cores containing the full histone octamer exhibit short-range mobility over 1O-bp DNA intervals. Thus the rotational setting of the DNA around the octamer is conserved during these nucleosome core movements (16). This temperature-dependent short-range mobility is distinct from the previously observed nucleosome sliding at relatively high ionic strengths (17, 18), which probably results from the weakening of histone-DNA interactions.Because of the probable importance of H1 as a general repressor, we have investigated the effect of the binding of linker histones on nucleosome mobility. Nucleosome positioning on sea urchin 58 rDNA is well characterized both without (19)(20)(21) and with (21) bound linker histones. We show that histones H1 and H5 (a member of the H1 family found in nucleated erythrocytes) effectively suppress the redistribution of histone octamers between possible nucleosome positions on this DNA. If nucleosome mobility is required for access to the DNA, then H1 could function as a repressor outside the context of the 30-nm fiber through its immobilization of nucleosome cores. MATERIALS AND METHODSPreparation of DNA Substrates. The 207-bp sea urchin 5S rDNA fragments were generated from the tandemly repeated insert of plasmid p5S207-18 (22) by Ava I restriction digestion. Head-to-tail dimers of this sequence were obtained by ligation at the asymmetric Ava I s...
BackgroundThe DNA methylation profiles of mammalian cell lines differ from those of the primary tissues from which they were derived, exhibiting increasing divergence from the in vivo methylation profile with extended time in culture. Few studies have directly examined the initial epigenetic and transcriptional consequences of adaptation of primary mammalian cells to culture, and the potential mechanisms through which this epigenetic dysregulation occurs is unknown.ResultsWe demonstrate that adaptation of mouse embryonic fibroblasts to cell culture results in a rapid reprogramming of epigenetic and transcriptional states. We observed global 5-hydroxymethylcytosine (5hmC) erasure within three days of culture initiation. Loss of genic 5hmC was independent of global 5-methylcytosine (5mC) levels and could be partially rescued by addition of vitamin C. Significantly, 5hmC loss was not linked to concomitant changes in transcription. Discrete promoter-specific gains of 5mC were also observed within seven days of culture initiation. Against this background of global 5hmC loss we identified a handful of developmentally important genes that maintained their 5hmC profile in culture, including the imprinted loci Gnas and H19. Similar outcomes were identified in the adaption of CD4+ T cells to culture.ConclusionsWe report a dramatic and novel consequence of adaptation of mammalian cells to culture in which global loss of 5hmC occurs, suggesting rapid concomitant loss of methylcytosine dioxygenase activity. The observed epigenetic and transcriptional re-programming occurs much earlier than previously assumed, and has significant implications for the use of cell lines as faithful mimics of in vivo epigenetic and physiological processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0576-y) contains supplementary material, which is available to authorized users.
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