Faculty Bibliography

Hematopoietic stem cells (HSCs) reside at the top of the hematopoietic hierarchy and are the origin of all blood cells produced throughout an individual's life. The balance between HSC self-renewal and differentiation is maintained by various intrinsic and extrinsic mechanisms. Among these, the molecular pathways that restrict cell cycle progression are critical to the maintenance of functional HSCs. Alterations in the regulation of cell cycle progression in HSCs invariably lead to the development of hematologic malignancies or bone marrow failure syndromes. Here we report that hematopoietic-specific genetic inactivation of Sin3B, an essential component of the mammalian Sin3-HDAC corepressor complex, severely impairs the competitive repopulation capacity of HSCs. Sin3B-deleted HSCs accumulate and fail to properly differentiate following transplantation. Moreover, Sin3B inactivation impairs HSC quiescence and sensitizes mice to myelosuppressive therapy. Together, these results identify Sin3B as a novel and critical regulator of HSC functions.

Cellular senescence is defined as a state of stable cell-cycle arrest that is distinct from quiescence and terminal differentiation. Many stimuli can induce senescence, including telomere shortening and oncogene activation. The phenotypes elicited by pro-senescent signals can be heterogeneous depending on the stimulus and the cell type affected. To date, there is not a definitive marker that can ubiquitously and specifically mark all senescent cells. Therefore, several independent markers must be utilized to ascertain the senescent state of a cell or group of cells. Here, we describe common assays used to assess oncogenic Ras-induced senescence.

URI is an unconventional prefoldin, RNA polymerase II interactor that functions as a transcriptional repressor, and is part of a larger nuclear protein complex. The components of this complex and the mechanism of transcriptional repression have not been characterized. Here we show that the KRAB-associated protein 1 (KAP1) and the protein phosphatase PP2A interact with URI. Mechanistically, we show that KAP1 phosphorylation is decreased following recruitment of PP2A by URI. We functionally characterize the novel URI-KAP1-PP2A complex, demonstrating a role of URI in retrotransposon repression, a key function previously demonstrated for the KAP1-SETDB1 complex. Microarray analysis of annotated transposons revealed a selective increase in the transcription of LINE-1 and L1PA2 retroelements upon knockdown of URI. These data unveil a new nuclear function of URI and identify a novel post-transcriptional regulation of KAP1 protein that may have important implications in reactivation of transposable elements in prostate cancer cells.

Perturbations in cell cycle regulators elicit hyperproliferation and the inability of cells to exit the cell cycle, which are common occurrences in cancer. Recently, we have demonstrated that Sin3B, an essential component of the mammalian histone deacetylase repressor complex, is required for oncogene-induced senescence both in vitro and in vivo. Surprisingly, primary Sin3B-null cells are not readily transformed upon ectopic expression of oncogenic Ras. Thus, these observations indicate that loss of Sin3B uncouples the ability to undergo senescence from the sensitivity to transformation in primary cells. To better understand the relationship between Sin3B, senescence and transformation, we have generated novel immortalized cell lines genetically inactivated for Sin3B. Our results indicate that in these Sin3B-deleted cells, expression of oncogenic Ras promotes transformation and provides a proliferative advantage. Interestingly, we have demonstrated that the paired amphipathic helix 2 (PAH2) domain is crucial for the tumor suppressive nature of Sin3B. To elucidate the molecular bases for these effects, immunoprecipitation and mass-spectrometry was performed to identify the proteins associated with Sin3B and its loss-of-function mutants. We have now identified novel interactors of the Sin3B complex and defined crucial domains responsible for the complex's assembly. These observations suggest that Sin3B functions as a non-classical tumor suppressor which serves to limit the transformative potential of cancer cells. The restoration of Sin3B functionality and downstream pathways may prove useful in the design of targeted therapies to treat advanced disease

Revolutionizing treatment strategies is an urgent clinical need in the fight against cancer. Recently the scientific community has recognized chromatin-associated proteins as promising therapeutic candidates. However, there is a need to develop more targeted epigenetic inhibitors with less toxicity. Sin3 family is one such target which consists of evolutionary conserved proteins with two paralogues Sin3A and Sin3B. Sin3A/B are global transcription regulators that provide a versatile platform for diverse chromatin-modifying activities. Sin3 proteins regulate key cellular functions that include cell cycle, proliferation, and differentiation, and have recently been implicated in cancer pathogenesis. In this chapter, we summarize the key concepts of Sin3 biology and elaborate the recent advancements in the role of Sin3 proteins in cancer with specific examples in multiple endocrine neoplasia type 2, pancreatic ductal adenocarcinoma, and triple negative breast cancer. Finally, a program to create an integrative approach for screening antitumor agents that target chromatin-associated factors like Sin3 is presented.

The role of senescence as a tumor suppressor is well established; however, recent evidence has revealed novel paracrine functions for senescent cells in relation to their microenvironment, most notably protumorigenic roles in certain contexts. Senescent cells are capable of altering the inflammatory microenvironment through the senescence-associated secretory phenotype, which could have important consequences for tumorigenesis. The role of senescent cells in a highly inflammatory cancer like pancreatic cancer is still largely undefined, apart from the fact that senescence abrogation increases tumorigenesis in vivo. This review will summarize our current knowledge of the phenomenon of cellular senescence in pancreatic ductal adenocarcinoma, its overlapping link with inflammation, and some urgent unanswered questions in the field.

The Polycomb group protein Bmi-1 is an essential regulator of cellular senescence and is believed to function largely through the direct repression of the Ink4a/Arf locus. However, concurrent deletion of Ink4a/Arf does not fully rescue the defects detected in Bmi-1(-/-) mice, indicating that additional Bmi-1 targets remain to be identified. The expression of the chromatin-associated Sin3B protein is stimulated by oncogenic stress, and is required for oncogene-induced senescence. Here we demonstrate that oncogenic stress leads to the dissociation of Bmi-1 from the Sin3B locus, resulting in increased Sin3B expression and subsequent entry into cellular senescence. Furthermore, Sin3B is required for the senescent phenotype and elevated levels of reactive oxygen species elicited upon Bmi-1 depletion. Altogether, these results identify Sin3B as a novel direct target of Bmi-1, and establish Bmi-1-driven repression of Sin3B as an essential regulator of cellular senescence.

Acetylation is correlated with chromatin decondensation and transcriptional activation, but its regulation by histone deacetylase (HDAC)-bearing corepressor complexes is poorly understood. Here, we describe the mechanism of assembly of the mammalian Sin3L/Rpd3L complex facilitated by Sds3, a conserved subunit deemed critical for proper assembly. Sds3 engages a globular, helical region of the HDAC interaction domain (HID) of the scaffolding protein Sin3A through a bipartite motif comprising a helix and an adjacent extended segment. Sds3 dimerizes through not only one of the predicted coiled-coil motifs but also, the segment preceding it, forming an approximately 150-A-long antiparallel dimer. Contrary to previous findings in yeast, Sin3A rather than Sds3 functions in recruiting HDAC1 into the complex by engaging the latter through a highly conserved segment adjacent to the helical HID subdomain. In the resulting model for the ternary complex, the two copies of the HDACs are situated distally and dynamically because of a natively unstructured linker connecting the dimerization domain and the Sin3A interaction domain of Sds3; these features contrast with the static organization described previously for the NuRD (nucleosome remodeling and deacetylase) complex. The Sds3 linker features several conserved basic residues that could potentially maintain the complex on chromatin by nonspecific interactions with DNA after initial recruitment by sequence-specific DNA-binding repressors.