Faculty Publications

Cellular senescence is an irreversible proliferation arrest, tumor suppression process and likely contributor to tissue aging. Senescence is often characterized by domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), which repress expression of proliferation-promoting genes. Given its likely contribution to tumor suppression and tissue aging, it is essential to identify all components of the SAHF assembly pathway. Formation of SAHF in human cells is driven by a complex of histone chaperones, namely, HIRA and ASF1a. In yeast, the complex orthologous to HIRA/ASF1a contains two additional proteins, Hpc2p and Hir3p. Using a sophisticated approach to search for remote orthologs conserved in multiple species through evolution, we identified the HIRA-associated proteins, UBN1 and UBN2, as candidate human orthologs of Hpc2p. We show that the Hpc2-related domain of UBN1, UBN2, and Hpc2p is an evolutionarily conserved HIRA/Hir-binding domain, which directly interacts with the N-terminal WD repeats of HIRA/Hir. UBN1 binds to proliferation-promoting genes that are repressed by SAHF and associates with histone methyltransferase activity that methylates lysine 9 of histone H3, a site that is methylated in SAHF. UBN1 is indispensable for formation of SAHF. We conclude that UBN1 is an ortholog of yeast Hpc2p and a novel regulator of senescence.

Integrated retroviral DNA is subject to epigenetic gene silencing, resulting in loss of expression of viral genes as well as reporter or therapeutic genes transduced by retroviral vectors. Possible mediators of such silencing include the histone deacetylase (HDAC) family of cellular proteins. We previously isolated HeLa cell populations that harbored silent avian sarcoma virus-based green fluorescent protein (GFP) vectors that could be reactivated by treatment with HDAC inhibitors. Here, we developed a small interfering RNA (siRNA)-based. approach to identify specific host factors that participate in the maintenance of silencing. Knockdown of HDAC1, the transcriptional repressor Daxx (a binding partner of HDAC1), or heterochromatin protein 1 gamma resulted in robust and specific GFP reporter gene reactivation. Analyses of cell clones and diverse GFP vector constructs revealed that the roles of HDAC1 and Daxx in retroviral silencing are largely independent of the integrati!

To perform structure/function analyses of a protein in vivo, ideally one should be able to simultaneously abolish expression of the endogenous wild-type protein, substitute it with a form of the protein containing a targeted mutation, and analyze the functional consequences. Until recently, this was a highly challenging and/or laborious approach in mammalian systems, requiring a targeted gene knockin in a human cell line or mouse. Herein is described a RNA interference (RNAi)-based approach to achieve this much more simply in mammalian cells. A single retrovirus has been constructed, which directs expression of a short hairpin RNA (shRNA) to knockdown expression of the endogenous protein of interest; a cDNA coding for a wild-type or mutant version of the same protein that also contains "silent mutations" that do not affect the protein sequence, but do make the mRNA resistant to the shRNA; and a puromycin-resistance gene to allow rapid drug selection of the virus-infected cells. Using this virus, expression of the endogenous Anti-Silencing Function 1a (ASF1a) histone chaperone has been efficiently replaced in primary human cells, by an ectopically expressed epitope-tagged version. Moreover, the virus is designed so that other shRNA and shRNA-resistant cDNA cassettes can easily be substituted, making the approach readily applicable to other protein targets.

Cellular information is encoded genetically in the DNA nucleotide sequence and epigenetically by the "histone code", DNA methylation and higher-order packaging of DNA into chromatin. Cells possess intricate mechanisms to sense and repair damage to DNA and the genetic code. However, nothing is known of the mechanisms, if any, that repair and/or compensate for damage to epigenetically encoded information, predicted to result from perturbation of DNA and histone modifications or other changes in chromatin structure. Here we show that primary human cells respond to a variety of small molecules that perturb DNA and histone modifications by recruiting HP1 proteins to sites of altered pericentromeric heterochromatin. This response is essential to maintain the HP1-binding kinetochore protein hMis12 at kinetochores and to suppress catastrophic mitotic defects. Recruitment of HP1 proteins to pericentromeres depends on histone H3.3 variant deposition, mediated by the HIRA histone chaperone. These data indicate that defects in pericentromeric epigenetic heterochromatin modifications initiate a dynamic HP1-dependent response that rescues pericentromeric heterochromatin function and is essential for viable progression through mitosis.

Senescence is characterized by an irreversible cell proliferation arrest. Specialized domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), are thought to contribute to the irreversible cell cycle exit in many senescent cells by repressing the expression of proliferation-promoting genes such as cyclin A. SAHF contain known heterochromatin-forming proteins, such as heterochromatin protein 1 (HP1) and the histone H2A variant macroH2A, and other specialized chromatin proteins, such as HMGA proteins. Previously, we showed that a complex of histone chaperones, histone repressor A (HIRA) and antisilencing function 1a (ASF1a), plays a key role in the formation of SAHF. Here we have further dissected the series of events that contribute to SAHF formation. We show that each chromosome condenses into a single SAHF focus. Chromosome condensation depends on the ability of ASF1a to physically interact with its deposition substrate, histone H3, in addition to its cochaperone, HIRA. In cells entering senescence, HP1 gamma, but not the related proteins HP1 alpha and HP1 beta, becomes phosphorylated on serine 93. This phosphorylation is required for efficient incorporation of HP1 gamma into SAHF. Remarkably, however, a dramatic reduction in the amount of chromatin-bound HP1 proteins does not delectably affect chromosome condensation into SAHF. Moreover, abundant HP1 proteins are not required for the accumulation in SAHF of histone H3 methylated on lysine 9, the recruitment of macroH2A proteins, nor other hallmarks of senescence, such as the expression of senescence-associated P-galactosidase activity and senescence-associated cell cycle exit. Based on our results, we propose a stepwise model for the formation of SAHF.

Cellular senescence is an irreversible proliferation arrest of primary cells and an important tumor suppression process. Senescence is often characterized by domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), which repress expression of proliferation-promoting genes. Formation of SAHF is driven by a complex of histone chaperones, HIRA and ASF1a, and depends upon prior localization of HIRA to PML nuclear bodies. However, how the SAHF assembly pathway is activated in senescent cells is not known. Here we show that expression of the canonical Wnt2 ligand and downstream canonical Wnt signals are repressed in senescent human cells. Repression of Wnt2 occurs early in senescence and independently of the pRB and p53 tumor suppressor proteins and drives relocalization of HIRA to PML bodies, formation of SAHF and senescence, likely through GSK3beta-mediated phosphorylation of HIRA. These results have major implications for our understanding of both Wnt signaling and senescence in tissue homeostasis and cancer progression.

In senescent cells, specialized domains of transcriptionally silent senescence-associated heterochromatic foci (SAHF), containing heterochromatin proteins such as HP1, are thought to repress expression of proliferation-promoting genes. We have investigated the composition and mode of assembly of SAHF and its contribution to cell cycle exit. SAHF is enriched in a transcription-silencing histone H2A variant, macroH2A. As cells approach senescence, a known chromatin regulator, HIRA, enters PML nuclear bodies, where it transiently colocalizes with HP1 proteins, prior to incorporation of HP1 proteins into SAHF. A physical complex containing HIRA and another chromatin regulator, ASF1a, is rate limiting for formation of SAHF and onset of senescence, and ASF1a is required for formation of SAHF and efficient senescence-associated cell cycle exit. These data indicate that HIRA and ASF1a drive formation of macroH2A-containing SAHF and senescence-associated cell cycle exit, via a pathway that appears to depend on flux of heterochromatic proteins through PML bodies.