The Laboratory of Elizaveta Benevolenskaya
at the University of Illinois at Chicago
at the University of Illinois at Chicago
The thought that one might understand mechanistically how cells decide whether to continue to proliferate or to differentiate has always been dreamlike to me. Cells that undergo uncontrolled proliferation or maintain poorly differentiated state predispose individuals to cancer. The knowledge of the critical molecular events that control the operating signal transduction pathways is necessary to understand cancer development. Yet, recent advances in molecular and computational biology have made possible to study, genome-wide, transcriptional regulatory networks, and availability of knockdown techniques reveals their relevance to cell growth and allows to put them in hierarchical order. By using both traditional methods and high-throughput approaches, we are studying mechanisms that orchestrate initiation and progression of differentiation.
My research plans will be aimed at understanding how, by positive or negative regulation of transcription, RBP2 and other pRB-interacting proteins affect differentiation. These studies will shed some light on how pRB cooperates with cell-fate determining transcription factors, which is a very poorly understood tumor suppressor function of pRB. If we reach global understanding of the central effectors of pRB involved in transcriptional regulatory interactions, we will know how to manipulate it to treat cancer.
The mechanism of mediating differentiation by RBP2
Inactivation of the retinoblastoma gene (RB) is viewed as a necessary step in the development of human cancers. It might be a result of a mutation in the RB gene or, more commonly, hyperphosphorylation of pRB . Both types of inactivation impair the ability of pRB to interact with some cellular proteins. These protein-protein interactions are believed to be responsible for several processes deregulated in cancer. The view of pRB as a tumor suppressor has been supported by its role in the negative regulation of cell cycle progression as well as the regulation of the E2F family of transcription factors. However, one of the pRB tumor suppressor functions is its ability to promote differentiation and senescence, a process during which the cell stops dividing and aquires a certain cell fate. There is a growing consensus that this might be the primary tumor suppressor function of pRB. Initially, we were focused on the search for proteins that mediate the ability of pRB to induce differentiation. We showed that the ability of pRB to promote differentiation correlates with its ability to bind and inhibit the RBP2 protein. Cells deficient in RBP2 have an enhanced ability to execute the differentiation program and override the requirement of pRB for terminal differentiation. Our studies are aimed on understanding the signaling pathway converging on pRB and RBP2. If we reach a global understanding of the central effectors of pRB involved in transcriptional regulatory interactions, we will know how to manipulate it to treat cancer.
Our studies suggest that in mammalian cells RBP2 (RBBP2/JARID1A) and pRB share a common, although sometimes opposing, role in the regulation of differentiation. Not only do they form protein complexes in vivo, but also act, at least in some cases directly, on the same target genes. Some of these targets, such as genes encoding BRD proteins, have been associated with cell fate determination, other targets, such as osteocalcin, have been connected to a specific stage of differentiation. RBP2 targets include, to a large extent, promoters of genes encoding mitochondrial proteins or DNA-binding proteins. Comparison of RBP2 occupancy at different stages of monocytic differentiation showed stage-specific distribution across both categories of genes and correlated with the occurrence of hematopoietic transcription factors. Recent discoveries in the enzymology of chromatin show that RBP2 possesses multiple signature motifs that potentially direct its binding to methylated chromatin. We are looking for the constituents of RBP2 complexes that are important for its effects on transcription. The results of our studies suggest that the interaction of pRB with RBP2 provides a general control over the cellular decision whether to withdraw from the cell cycle and differentiate.
RBP2 family proteins have been associated with human malignancies. While a RBP2 gene translocation has been described in a child with acute myeloid leukemia, the RBP2 homolog PLU-1 is a cancer specific antigen overexpressed in the majority of breast cancer cases. We are currently developing RBP2 and PLU-1 mice models that will be useful to study the role of RBP2 protein family in pRB-mediated differentiation and in cellular transformation.
A combination of GeneAtlas, Oncomine analysis and bioinformatics analysis of CGH data indicates that deletions in the RBP2 locus, correlating with downregulation of RBP2 level and upregulation of RBP2 target gene expression occur in a statistically significant number of hematopoietic malignancies including lymphoma and more specifically Diffuse Large B-Cell Lymphoma (DLBCL), the most common adult lymphoid malignancy. Oncogene analysis has shown that the transcriptional signatures for the 3 DLBCL subtypes represent a high number of RBP2 target genes. We are studying the expression level of RBP2 and its target genes in tumor tissues with the aim of establishing a characteristic expression signature of leukemia and lymphoma cells.
Control of gene expression by PLU1, the closest RBP2 homolog
KDM5B/JARID1B/PLU1 is the RBP2 family member associated with malignancy. We study if its depletion in a breast cancer line promotes differentiation. We are also investigating correlation of PLU1 binding with the appearance of tri-, di-, mono- and unmethylated H3K4 as well as with gene repression or activation.
The role of RBP2 and JARID2 in cardiomyocytes
Fetal and neonatal cardiac myocytes are able to proliferate, however this ability decreases progressively with an organism's age such that adult cardiomyocytes are unable to divide. A major problem arising from the inability of cardiomyocytes to proliferate is that the mature heart is unable to regenerate new tissue following severe injury. JARID2/JMJ has been originally described as an important factor in heart development. Recently, JARID2 was identified as a key mediator of H3K9 and H3K27me3 marks through interaction with methyltransferases, G9a and GLP, and PRC2, respectively. We study which genes are targeted by RBP2 and JARID2 during cardiac myocyte growth and whether they are subject of histone demethylation.
Beshiri, M.L., K.B. Holmes, W.F. Richter, S. Hess, A.B.M.M.K. Islam and E. V. Benevolenskaya. KDM5A regulates H3K4 methylation contributing to repression of cell cycle genes during differentiation. Submitted.
Islam, A.B.M.M.K., W.F. Richter, L. Jakobs, N. Lopez-Bigas and E.V. Benevolenskaya. Coregulation of histone-modifying enzymes in cancer. Submitted.
Abul BMMK Islam, William F. Richter, Nuria Lopez-Bigas1 and Elizaveta V. Benevolenskaya. Selective targeting of histone methylation.. Cell Cycle 10:3, 413-424; February 1, 2011.
Nicolay, B.N., B. Bayarmagnai, N.S. Moon, E.V. Benevolenskaya and M.V. Frolov. 2010. Combined inactivation of pRB and Hippo pathways induces dedifferentiation in the Drosophila retina. PLoS Genetics 6(4): e1000918.
Beshiri ML, Islam A, DeWaal DC, Richter WF, Love J, Lopez-Bigas N, Benevolenskaya EV. Genome-wide analysis using ChIP to identify isoform-specific gene targets. J Vis Exp. 2010 Jul 7;(41). pii: 2101. doi: 10.3791/2101.
Also see video at: http://www.jove.com/index/Details.stp?ID=2101
Lopez-Bigas N, Kisiel TA, Dewaal DC, Holmes KB, Volkert TL, Gupta S, Love J, Murray HL, Young RA, Benevolenskaya EV. Genome-wide analysis of the H3K4 histone demethylase RBP2 reveals a transcriptional program controlling differentiation. Mol Cell. 2008 Aug 22;31(4):520-30.
Benevolenskaya, E.V., H.L. Murray, P. Branton, R.A. Young, and W.G. Kaelin, Jr.. 2005. Binding of pRB to the PHD protein RBP2 promotes cellular differentiation. Molecular Cell 18: 623-635.
E.V. Benevolenskaya. 2007.Histone H3K4 demethylases are essential in development and differentiation. Biochem. Cell Biol. Review. 85: 435-443.
Serebriiskii, I.G., O. Mitina, E.N. Pugacheva, E.V. Benevolenskaya, E. Kotova, G.G. Toby, V. Khazak, W.G. Kaelin, J. Chernoff, and E.A. Golemis. 2002. Detection of peptides, proteins, and drugs that selectively interact with protein targets. Genome Research 12: 1785-1791.