Histone H3.3 is a constitutively-expressed variant of the H3 histones that is incorporated into chromatin through replication-independent mechanisms. H3.3 also plays critical roles in genome regulation and epigenetic inheritance. HIRA is required for H3.3 deposition into euchromatin. The H3.3-specific chaperone, DAXX, together with the chromatin remodeling factor, ATRX, regulate H3.3 incorporation into heterochromatin. DAXX and ATRX are also components of promyelocytic nuclear bodies (PML-NBs), which have been identified as sites of H3.3 chromatin assembly and linked to transcriptional silencing, DNA damage, senescence, and anti-viral and stress responses. Recently, specific driver-point mutations in H3.3 were identified in pediatric high-grade gliomas (HGGs) and skeletal cancers. Loss-of-function mutations in DAXX and ATRX have also been identified in pancreatic neuroendocrine tumors and brain cancer, which suggests that proper regulation of H3.3 chromatin assembly is essential for preventing tumorigenesis. Although H3.3 is highly conserved, all of the mechanisms that regulate its functions have not been fully elucidated. This thesis describes two studies that identify novel H3.3 regulatory mechanisms, which have important implications for understanding epigenetic inheritance and disease pathogenesis.
1) The RNase P subunit, Rpp29, regulates histone H3.3-mediated functions in transcription and epigenetic inheritance. We previously reported that Rpp29 represses H3.3 incorporation into heterochromatin. Here, we identify a sequence element in the eukaryote-specific N-terminus of Rpp29 that constitutes the H3.3-interaction domain. Since the oncogenic H3.3-point mutations alter the interaction with Rpp29, it suggests that they could dysregulate Rpp29 function in chromatin assembly. We also show that Rpp29 represses H3.3 incorporation into single-copy, rRNA, and tRNA genes as well as the expression of both mRNA and the corresponding protein. Additionally, Rpp29 promotes the deposition of histone post-translational modifications (PTMs) associated with transcriptional silencing (i.e. H3K9me3 and H3K27me3) and represses PTMs associated with transcriptional activation. Taken together, these data suggest that Rpp29 is a genome-wide regulator of H3.3-mediated transcriptional and epigenetic regulation.
2) PML is recruited to heterochromatin during S phase to coordinate histone H3.3 chromatin assembly with DNA replication. Here, we use a DAXX-ATRX-H3.3-regulated transgene array that can be visualized in single-living cells to investigate the function of PML-NBs in heterochromatin regulation. SUMOylation is required for PML-NB factors (e.g. DAXX and PML) to be recruited to the transgene array and for H3.3 to be incorporate, which suggests that PML-NBs function to maintain the H3.3-mediated silencing mechanism at heterochromatin sites. We also found that PML represses H3.3 incorporation and promotes H3K9me3 deposition. Since PML is recruited the transgene array during S phase, it suggests that it represses DAXX-ATRX-mediated H3.3 chromatin assembly during DNA synthesis. Additionally, DAXX and ATRX repress the PML accumulation at the array, which suggests that DAXX and ATRX also regulate PML function in H3.3 chromatin assembly by restricting its recruitment to S phase.
In summary, these studies reveal that histone H3.3 chromatin assembly is highly integrated with transcriptional, RNA processing, nuclear organizational, and cell cycle-regulatory mechanisms. Elucidating the critical steps in H3.3-mediated epigenetic inheritance will have important implications for understanding development, viral latency, and cancer.
|School:||University of the Sciences in Philadelphia|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 80/07(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Oncology|
|Keywords:||Epigenetics, Histone h3.3, Pml, Replication, Rnase p, Transcription|
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