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The histone variant H3.3 is involved in diverse biological processes, including development, transcriptional memory and transcriptional reprogramming, as well as diseases, including most notably malignant brain tumors. Recently, we developed a knockout mouse model for the H3f3b gene, one of two genes encoding H3.3. Here, we show that targeted disruption of H3f3b results in a number of phenotypic abnormalities, including a reduction in H3.3 histone levels, leading to male infertility, as well as abnormal sperm and testes morphology. Additionally, null germ cell populations at specific stages in spermatogenesis, in particular spermatocytes and spermatogonia, exhibited increased rates of apoptosis. Disruption of H3f3b also altered histone post-translational modifications and gene expression in the testes, with the most prominent changes occurring at genes involved in spermatogenesis. Finally, H3f3b null testes also exhibited abnormal germ cell chromatin reorganization and reduced protamine incorporation. Taken together, our studies indicate a major role for H3.3 in spermatogenesis through regulation of chromatin dynamics.

Background The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b. Results H3f3b knockout (KO) mice exhibit a semilethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in mouse embryonic fibroblasts (MEFs) with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile. Conclusions Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development.

Myc and N-Myc have widespread impacts on the chromatin state within cells, both in a gene-specific and genome-wide manner. Our laboratory uses functional genomic methods including chromatin immunoprecipitation (ChIP), ChIP-chip, and, more recently, ChIP-seq to analyze the binding and genomic location of Myc. In this chapter, we describe an effective ChIP protocol using specific validated antibodies to Myc and N-Myc. We discuss the application of this protocol to several types of stem and cancer cells, with a focus on aspects of sample preparation prior to library preparation that are critical for successful Myc ChIP assays. Key variables are discussed and include the starting quantity of cells or tissue, lysis and sonication conditions, the quantity and quality of antibody used, and the identification of reliable target genes for ChIP validation.

Myc proteins have long been modeled to operate strictly as classic gene-specific transcription factors; however, we find that N-Myc has a robust role in the human genome in regulating global cellular euchromatin, including that of intergenic regions. Strikingly, 90% to 95% of the total genomic euchromatic marks histone H3 acetylated at lysine 9 and methylated at lysine 4 is N-Myc–dependent. However, Myc regulation of transcription, even of genes it directly binds and at which it is required for the maintenance of active chromatin, is generally weak. Thus, Myc has a much more potent ability to regulate large domains of euchromatin than to influence the transcription of individual genes. Overall, Myc regulation of chromatin in the human genome includes both specific genes, but also expansive genomic domains that invoke functions independent of a classic transcription factor. These findings support a new dual model for Myc chromatin function with important implications for the role of Myc in cancer and stem cell biology, including that of induced pluripotent stem cells. [Cancer Res 2008;68(23):9654–62]

MeCP2 is a member of a family of proteins [methyl- (cytosine-guanine)CpG-binding proteins] that bind specifically to methylated DNA and induce chromatin remodeling and gene silencing. Dietary deficiency of folate, choline and methionine causes decreased tissue S-adenosylmethionine concentrations (methyl deficiency), global DNA hypomethylation, hepatic steatosis, cirrhosis and ultimately hepatic tumorigenesis in rodents. We investigated the effects of this diet on expression of MeCP2 during pre-neoplastic transformation of liver tissue. After 9 weeks, MeCP2 mRNA level was slightly higher in methyl-deficient rats compared with replete controls, while after 36 weeks, a difference in MeCP2 mRNA level was no longer observed. In contrast, MeCP2 protein level was reduced almost 2-fold in the deficient rats compared with replete controls at both 9 and 36 weeks. Conversely, a second methyl-CpG-binding protein, MBD2, showed increased levels of both message and protein at the two time points. Low MeCP2 protein in the deficient rats was associated with a low level of the co-repressor protein, Sin3a, at 36 weeks. Moreover, a known gene target of MeCP2, the tumor suppressor gene metallothionein-I, was over-expressed in the deficient rat livers at both 9 and 36 weeks, suggesting that reduction in MeCP2 may have functional consequences. Methyl deficiency also caused an increase in the ratio of long to short variants of MeCP2 transcripts. This finding suggests that reduced MeCP2 protein level is the result of a reduced rate of translation. Reduction of MeCP2 protein expression may influence the initiation and/or progression of hepatic cancer induced by methyl deficiency and may provide a useful marker of pre-neoplastic change.