Stem Cell Regulation by Alternative Splicing

Stem Cell Regulation by Alternative Splicing Daniel Scacalossi Introduction Pluripotent embryonic stem cells possess genomic regulatory systems which are of interest to study for their unique ability to become any cell type in an organism. Research is advancing our understanding of the mechanisms in which pluripotency is maintained in embryonic stem cells (ESCs) and how this state can be induced in somatic cells producing induced pluripotent stem cells (iPSCs). It has been confirmed that the transcription factors Oct4 and Sox2, Klf4 and c-Myc reprogram somatic cells to iPSCs. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663–676. However, the ability of iPSCs to regenerate and differentiate is not equivalent to ESC potential. iPS cells undergo senescence and apoptosis after short periods in culture. Further, iPS cells prove difficult when prompted to form a full organism. Although some lab groups are able to succeed at this, it is at a much lower efficiency than ES cells. Vogel G., Reprogrammed Cells Come Up Short, for Now. Science 5 March 2010: Vol. 327 no. 5970 p. 1191 DOI: 10.1126/science.327.5970.1191 The reasons are not known. However, epigenetic alterations may be involved. In 2010 Koche et al found that key histone modifications were modulated in the early moments of reprogramming. Of particular interest was the genome wide modulation of the H3K4me2 mark. Also among the aspects of gene regulation which are not well understood is the role of alternative splicing (AS) in maintaining pluripotency of stem cells. This review will consider epigenetic modifications and the implications of tissue specific AS with respect to genes such as oct4 which is known to regulate pluripotency via a specific protein isoform. The process of alternative splicing in eukaryotes is credited with facilitating a vast diversity of proteins from a finite genome. Approximately 90% of human RNA is alternatively spliced. Eric T. Wang1,2,7, Rickard Sandberg1,3,7, Shujun Luo4, Irina Khrebtukova4, Lu Zhang4, Christine Mayr5, Stephen F. Kingsmore6, Gary P. Schroth4 & Christopher B. Burge: Alternative isoform regulation in human tissue transcriptomes. Nature 2008. However, alternative splicing could be responsible for some unexpected cellular processes. An alternative splicing event has recently been implicated in ESC pluripotency. An understanding of how splicing events are regulated will help to contextualize the role of alternative splicing in ESC maintenance. Regulation of Alternate Splicing by PTB Perhaps the most well-known model of pre-mRNA splicing comes from studies of somatic sex determination in Drosophila melanogaster. In this model the Sex lethal gene (Sxl) is expressed in the female fly, repressing splice variants that give rise to male phenotypic development. Sxl protein directly competes with splicing factor (U2AF) for its RNA binding site. Sosnowski BA, Belote JM, McKeownM. Sex-specific regulation of the male-specific lethal-1 dosage compensation gene in drosophila 1989. Cell 58:449–59 It is essential that U2AF bind the polypyrimidine tract of the pre-mRNA in order to recruit spliceosomal proteins needed for a splicing event. This is one of the more direct models illustrating regulation by non-spliceosomal proteins. Further studies have shown that the likelihood of generating one isoform verses another will be determined by multiple factors. The binding of intronic or exonic splicing enhancers (ISEs, ESEs) by serine/arginine-rich (SR) proteins facilitates AS events. Wang, Z; Burge, Cb. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA,  14: 802-813 (2008) ISEs and ESEs (and their suppressing counterparts) are cis-regulatory elements that recruit sequence-specific RNA-binding protein factors that activate (or repress) adjacent splice sites. The proteins recruited by these splicing regulatory elements facilitate or inhibit the formation of the spliceosome. However, because the specific alternative splicing result of a pre-mRNA transcript is essentially a competition between inclusion/exclusion of an exon, any mechanism that alters the rate of spliceosome assembly can act as a regulatory mechanism.  Black, Douglas L. Mechanisms of alternative pre-messenger RNA splicing. Annual Reviews of Biochemistry 72, (2003). Luco RF, Misteli T. More than a splicing code: integrating the role of RNA, chromatin and non-coding RNA in alternative splicing regulation. Curr Opin Genet Dev 2011 Aug;21(4):366-72. Epub 2011 Apr 15. PTB Recruited by Histone modifications Current opinions on alternative splicing regulation are that epigenetic marks can have an impact on the likelihood of which protein isoform is produced. Nucleosome enrichment at exons could be slowing down transcription elongation significantly so that splicing factors have more time to bind to and assemble at an exon that might otherwise be the less likely candidate due to its weaker binding affinity. This mechanism would apply to cases in which transcription and splicing occur concomitantly which is believed to be the majority of the time. Histone marks may function to control nucleosome density in a region of DNA where AS events occur thus affecting the rate of RNA pol II elongation. 7 These mechanisms are representative of AS regulation via modulation of RNA pol II kinetics. However, chromatin-binding adaptor protein MORF-related gene 15 (MRG15) has been implicated as a direct molecular link between the splicing factors and chromatin.9 Key histone modifications (H3K36me3 and others) are read by MRG15, selecting for the final splicing product via a polypyrimidine tract–binding protein (PTB) –dependent mechanism. A target for spliceosome assembly, the polypyrimidine tract is a pyrimidine rich region 15-20 base pairs in length. PTB has been observed to compete with the essential splicing factor U2AF for the binding of the polypyrimidine tract. PTB binding results in exclusion of an exon that would otherwise be included. PTB is therefore known as an AS regulatory protein. R. P. Carstens, E. J. Wagner, M. A. Garcia-Blanco An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract binding protein. Mol. Cell Biol, 2000 Oct;20(19):7388-400. It is thought that since pre-mRNA generally has a low affinity for PTB that H3-K36me3 binds MRG15 which aids in the recruitment of PTB to PTB-recognition sites on pre-mRNA. Reini F. Luco, Qun Pan, Kaoru Tominaga, Benjamin J. Blencowe, Olivia M. Pereira-Smith and Tom Misteli, Regulation of Alternative Splicing by Histone Modifications. Science 19 February 2010 Given that multiple histone modifications could lead to the recruitment of multiple chromatin-binding adaptor proteins, the possibilities for the regulation of splicing factors via epigenetic modifications are vast. Stem Cell Regulation by Alternate Splicing Current alternative splicing research has been uncovering ways in which AS events function to regulate stem cell differentiation. There are protein isoforms that have been implicated in the direction of ES cell differentiation. Among them is the OCT4A protein. The primary isoform involved in embryonic stem cell pluripotency is OCT4A which resides in the nucleus. OCT4B is the translation of a splicing event that skips exon 1 shortening the N terminus. X. Wang and J. Dai, Concise review: isoforms of OCT4 contribute to the confusing diversity in stemcell biology, Stem Cells, vol. 28, no. 5, pp. 885–893, 2010. J. Lee, H. K. Kim, J. Y. Rho, Y.M. Han, and J. Kim, The human OCT-4 isoforms differ in their ability to confer self-renewal, Journal of Biological Chemistry, vol. 281, no. 44, pp. 33554– 33565, 2006. G. Cauffman, H. van de Velde, I. Liebaers, and A. van Steirteghem, Oct-4 mRNA and protein expression during human preimplantation development, Molecular Human Reproduction, vol. 11, no. 3, pp. 173–181, 2005. Interestingly, although both isoforms contain a nuclear localization signal, only the OCT4A isoform is localized to the nucleus. FOXP1 It is well known that the Forkhead box (FOX) transcription factors regulate vast numbers of genes involved in developmental processes, differentiation and proliferation. Wijchers, P.J., Burbach, J.P., and Smidt, M.P. (2006). In control of biology: of mice, men and Foxes. Biochem. J. 397, 233–246. A recent study of FOXP1 has identified a highly conserved alternative splicing event that modulates transcriptional regulatory networks. Mathieu Gabut, Payman Samavarchi-Tehrani, Xinchen Wang, Valentina Slobodeniuc, Dave O’Hanlon, Hoon-Ki Sung, Manuel Alvarez, Shaheynoor Talukder, Qun Pan, Esteban O. Mazzoni, Stephane Nedelec, Hynek Wichterle, Knut Woltjen, Timothy R. Hughes, Peter W. Zandstra, Andras Nagy, Jeffrey L. Wrana, and Benjamin J. Blencowe An Alternative Splicing Switch Regulates Embryonic Stem Cell Pluripotency and Reprogramming Cell, Volume 147, Issue 1, 132-146, 15 September 2011 These experiments show that the inclusion of FOXP1 exon 18b is specific to self-renewing pluripotent hESCs and that the corresponding protein isoform serves to activate transcription of genes that function in embryonic cell pluripotency networks while suppressing genes that are associated with organ development, system development, and cell differentiation. The other major alternative splicing product includes FOXP1 exon 18 producing an mRNA that codes for a transcription factor that upregulates genes necessary for differentiation such as organ morphogenesis, receptor binding, multicellular organismal development and anatomical structure morphogenesis. The study examines differentiation-associated genes (GAS1, CITED2, WNT1, HESX1, BIK, and SFRP4) and observed that the canonical form of FOXP1 upregulates these genes. Importantly, the ESC-specific isoform FOXP1-ES upregulates expression of genes required for pluripotency: OCT4, NANOG, NR5A2, and GDF3. The mechanisms that regulate this newly discovered FOXP1 AS event are not known. The modulation of histone modifications during transition from stem cell towards differentiation is a curious phenomenon to consider as this process is known to have a profound impact on transcriptional activation and silencing. The findings of Luco et al of MRG15 acting as an adaptor between a histone mark and splicing regulator PTB, taken with AS events of FOXP1 suggests there is correlation between these processes. Koche et al dicovered genome wide modulation of the H3K4me2 mark which could play a role in mechanism similar to the Luco et al finding. Significance and going forward These results suggest that this AS event acts as a switch turning on programs of cell differentiation in hESCs. Understanding the factors that determine which mRNA results from FOXP1 splicing would be a step towards a comprehensive understanding of hESC maintenance. Given that ESCs are characterized as having a unique chromatin landscape and distinct epigentic marks (e.g. “bivalent” marks), one could conceive of a set of experiments in which the function of these marks over FOXP1 splicing is examined. For instance, a comparative mapping a set of histone modifications across the alternatively spliced FOXP1 region using quantitative chromatin immunoprecipitation in hESCs and differentiated cell types. Determining whether or not either or both of the mutually exclusive FOXP1 exons are PTB-dependent may also lead to an understanding of what factors govern FOXP1 splicing outcome. Histone marks have been shown to direct the outcome of AS events through H3-K36me3/MRG15–mediated recruitment of PTB.8 A Chromatin immunoprecipitation of MRG15 along FOXP1 could be a plausible way to test this theory. If this hypothesis were correct, one might expect overexpression of the H3-K36 methyltransferase to alter the dynamics of the inclusion of the PTB-dependent FOXP1 exon(s). Another possible protein of interest could be the Sgf29 chromatin binding protein. Earlier this year Bian et al showed that Sgf29 selectively binds H3K4me2/3 marks. In the mechanism elucidated by Bian et al Sgf29 recruits Spt-Ada-Gcn5 acetyltransferase (SAGA) complex. As mentioned in the introduction H3K4me2 levels undergo rapid dynamics in early reprogramming of stem cells. If Sgf29 were to act as a recruiter of PTB similar to MRG15 this might explain some of the changes in splicing products and subsequent modulation of transcriptional networks that have been observed in cell differentiation and reprogramming. FOXP1 splicing controls two distinct transcriptional pathways. The pluripotent master controller OCT4 is upregulated by the embryonic stem cell specific isoform suggests that the significance of the FOXP1 switch is profound. Deciphering the mechanisms that regulate an alternate splicing event as critical would play a substantial part to further our understanding of stem cell control. PAGE \* MERGEFORMAT 2 | Page