Bingying Zhou

INO80 Facilitates Pluripotency Gene Activation in Embryonic Stem Cell Self-Renewal, Reprogramming, and Blastocyst Development

The master transcription factors play integral roles in the pluripotency transcription circuitry of embryonic stem cells (ESCs). How they selectively activate expression of the pluripotency network while simultaneously repressing genes involved in differentiation is not fully understood. Here, we define a requirement for the INO80 complex, a SWI/SNF family chromatin remodeler, in ESC self-renewal, somatic cell reprogramming, and blastocyst development. We show that Ino80, the chromatin remodeling ATPase, co-occupies pluripotency gene promoters with the master transcription factors, and its occupancy is dependent on OCT4 and WDR5. At the pluripotency genes, Ino80 maintains an open chromatin architecture and licenses recruitment of Mediator and RNA polymerase II for gene activation. Our data reveal an essential role for INO80 in the expression of the pluripotency network and illustrate the coordination among chromatin remodeler, transcription factor, and histone-modifying enzyme in the regulation of the pluripotent state.

The THO complex regulates pluripotency gene mRNA export and controls embryonic stem cell self-renewal and somatic cell reprogramming.

Embryonic stem cell (ESC) self-renewal and differentiation are governed by a broad-ranging regulatory network. Although the transcriptional regulatory mechanisms involved have been investigated extensively, posttranscriptional regulation is still poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5 and is required for self-renewal at least in part by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, leading to decreased expression of pluripotency proteins that facilitates exit from self-renewal. THO is also important for the establishment of pluripotency, because its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicate that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and therefore uncover a role for this aspect of posttranscriptional regulation in stem cell fate specification.

INO80 governs superenhancer-mediated oncogenic transcription and tumor growth in melanoma

Superenhancers (SEs) are large genomic regions with a high density of enhancer marks. In cancer, SEs are found near oncogenes and dictate cancer gene expression. However, how oncogenic SEs are regulated remains poorly understood. Here, we show that INO80, a chromatin remodeling complex, is required for SE-mediated oncogenic transcription and tumor growth in melanoma. The expression of Ino80, the SWI/SNF ATPase, is elevated in melanoma cells and patient melanomas compared with normal melanocytes and benign nevi. Furthermore, Ino80 silencing selectively inhibits melanoma cell proliferation, anchorage-independent growth, tumorigenesis, and tumor maintenance in mouse xenografts. Mechanistically, Ino80 occupies >90% of SEs, and its occupancy is dependent on transcription factors such as MITF and Sox9. Ino80 binding reduces nucleosome occupancy and facilitates Mediator recruitment, thus promoting oncogenic transcription. Consistently, genes co-occupied by Ino80 and Med1 are selectively expressed in melanomas compared with melanocytes. Together, our results reveal an essential role of INO80-dependent chromatin remodeling in SE function and suggest a novel strategy for disrupting SEs in cancer treatment.

Protein Kinase CK2α Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2α Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma

The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies.

Histone Variant H2A.Z is Required for the Maintenance of Smooth Muscle Cell Identity as Revealed by Single-Cell Transcriptomics.

Background: Histone variants endow chromatin with specific structures, and play essential roles in development and diseases. However, little is known about their roles in controlling cell identity in vascular diseases. Methods: Given the cell heterogeneity in atherosclerotic lesions, we applied single-cell RNA-Sequencing to analyze diseased human arteries, and identified histone variant H2A.Z as a key histone signature to maintain vascular smooth muscle cell (VSMC) identity. Results: We show that H2A.Z occupies genomic regions near VSMC marker genes, and its occupancy is decreased in VSMCs undergoing dedifferentiation. Mechanistically, H2A.Z occupancy preferentially promotes nucleosome turnover, and facilitates the recruitment of SMAD3 and MED1, thereby activating VSMC marker gene expression. In addition, H2A.Z expression is dramatically reduced at both mRNA and protein levels in diseased human vascular tissues compared to those in normal arteries. Notably, in vivo overexpression of H2A.Z rescues injury-induced loss of VSMC identity and neointima formation. Conclusions: Together, our data introduce dynamic occupancy of a histone variant as a novel regulatory basis contributing to cell fate decisions, and imply H2A.Z as a potential intervention node for vascular diseases.

Cnot3 enhances human embryonic cardiomyocyte proliferation by promoting cell cycle inhibitor mRNA degradation

Uncovering the molecular basis of mammalian cardiomyocyte proliferation may eventually lead to better approaches for heart regeneration. Compared to extensively-studied transcriptional regulation, the roles of posttranscriptional regulation in cardiac cell fate decisions remain largely unknown. Here, we identified Cnot3 as a critical regulator in cardiomyocyte proliferation at the late stage of cardiac differentiation from human ESCs. Cnot3 was highly expressed in cardiomyocytes with higher proliferation potential in both human and mouse, and its depletion resulted in significant reduction in the proliferative capacity of cells. Furthermore, Cnot3 overexpression greatly enhanced proliferation in both cultured human cardiomyocytes and infarcted murine hearts. Mechanistically, the Ccr4-Not complex preferentially interacted with anti-proliferation gene transcripts in a Cnot3-dependent manner, and promoted their degradation. Together, our study supported the model that Cnot3 enhances cardiomyocyte proliferation by promoting cell cycle inhibitor mRNA degradation. It revealed a previously unrecognized role of mRNA degradation in cardiomyocyte growth, and suggested a potential strategy to control cardiac cell fates in development and diseases.

Posttranslational Modifications of Small G Proteins

The numerous biological functions of Ras superfamily small GTPases are highly dependent upon specific posttranslational modifications that guide their subcellular localization and interaction with regulators and effectors. Canonical modifications of their carboxyl termini include prenylation by farnesyl or geranylgeranyl isoprenoid lipids (Ras, Rho, Rab families). These serve as important components of their membrane targeting motifs and promote membrane binding, analogously to the cotranslational amino-terminal myristoylation of Arf family proteins. Reversible carboxymethylation of the prenylated cysteines and reversible acylation by one or more nearby palmitates promote dynamic membrane interactions to complement the permanent lipid modifications. Small GTPases are also regulated in both normal and disease states by several dynamic non-lipid posttranslational modifications. For example, many Ras and Rho family members are phosphorylated in an isoform-specific manner, largely by a select group of serine/threonine kinases such as protein kinase Cα or protein kinase A. Such phosphorylation events, as well as other modifications such as nitrosylation, mono- and di-ubiquitination, peptidyl-prolyl isomerization, acetylation, and oxidation, typically alter small GTPase location and/or interaction with regulatory molecules. By contrast, several distinct E3 ligases posttranslationally regulate small GTPase abundance and function at distinct cellular sites by promoting polyubiquitination and subsequent proteasomal degradation. Finally, numerous pathogenic bacterial toxins disrupt or enhance small GTPase function by a wide variety of posttranslational modifications including ADP ribosylation for which the Arf proteins are named. Here we summarize the rapidly evolving understanding of this fascinating area of small G protein regulation.

Abstract LB-217: CK2 protein kinase promotes resistance to MAPK pathway inhibition

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Small molecule kinase inhibitors have opened potential new avenues for treating cancers dependent on the RAS-RAF-MEK-ERK MAPK pathway, yet identification of both de novo/innate/intrinsic and acquired resistance mechanisms will be critical for the successful application of these inhibitors in the clinic. We interrogated the kinome to identify resistance mechanisms towards the novel ERK1/2-selective inhibitor SCH772984. We first utilized a kinome-focused RNAi screen to identify genes that, when silenced, sensitized KRAS-dependent pancreatic cancer cells to SCH772984. In our drug dose-response screen of 711 kinases (QIAGEN library), we used 4 independent siRNA duplexes to knock down each gene, treated at 5 different drug doses, then evaluated viability with a standard CellTiter-Glo assay. Nineteen kinases enhanced sensitivity to SCH772984 (where at least 2 siRNAs for each target decreased IC50) at least 5-fold, indicating that they could drive ERK1/2 inhibitor resistance. Among these were 4 of the 9 protein kinases (Cot, Raf-1, PAK3 and PRKCH) identified in a cDNA expression screen for kinases that caused resistance of BRAF-mutant melanoma to the BRAF inhibitor vemurafenib. We therefore hypothesized that at least some of the kinases that scored positive in this screen might be conserved both across the MAPK pathway and across different cancer types. The tetrameric protein kinase CK2 (formerly casein kinase II) has crucial roles in cell survival, proliferation and differentiation, and its expression and/or activity is dysregulated in cancers including melanoma. In very recent studies, we have now determined that the alpha subunit of CK2 is sufficient to cause resistance to each of the three ERK MAPK pathway inhibitors currently approved for treatment of melanoma: vemurafenib (BRAFi), dabrafenib (BRAFi) and trametinib (MEKi). Co-inhibition of the MAPK pathway and CK2 (e.g., with CX-4945, currently in phase I trials) further enhanced sensitivity. Thus, this combination may offer an effective regimen to forestall or delay tumor relapse. We also observed that CK2alpha maintains ERK phosphorylation in the presence of MAPK inhibitors, and that it posttranslationally reduces abundance of the ERK phosphatase DUSP6/MKP-3, in a kinase-dependent manner. The relationship of these findings to CK2-mediated mechanisms of drug resistance will be discussed. Citation Format: Bingying Zhou, Michelle Kassner, Holly Yin, Channing J. Der, Adrienne D. Cox. CK2 protein kinase promotes resistance to MAPK pathway inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-217. doi:10.1158/1538-7445.AM2014-LB-217