Sunil K. Mallanna

Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression. These small, non-coding RNAs are believed to regulate more than a third of all protein coding genes, and they have been implicated in the control of virtually all biological processes, including the biology of stem cells. The essential roles of miRNAs in the control of pluripotent stem cells were clearly established by the finding that embryonic stem (ES) cells lacking proteins required for miRNA biogenesis exhibit defects in proliferation and differentiation. Subsequently, the function of numerous miRNAs has been shown to control the fate of ES cells and to directly influence critical gene regulatory networks controlled by pluripotency factors Sox2, Oct4, and Nanog. Moreover, a growing list of tissue-specific miRNAs, which are silenced or not processed fully in ES cells, has been found to promote differentiation upon their expression and proper processing. The importance of miRNAs for ES cells is further indicated by the exciting discovery that specific miRNA mimics or miRNA inhibitors promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Although some progress has been made during the past two years in our understanding of the contribution of specific miRNAs during reprogramming, further progress is needed since it is highly likely that miRNAs play even wider roles in the generation of iPS cells than currently appreciated. This review examines recent developments related to the roles of miRNAs in the biology of pluripotent stem cells. In addition, we posit that more than a dozen additional miRNAs are excellent candidates for influencing the generation of iPS cells as well as for providing new insights into the process of reprogramming.

Elevating the levels of Sox2 in embryonal carcinoma cells and embryonic stem cells inhibits the expression of Sox2:Oct-3/4 target genes

Recent studies have identified large sets of genes in embryonic stem and embryonal carcinoma cells that are associated with the transcription factors Sox2 and Oct-3/4. Other studies have shown that Sox2 and Oct-3/4 work together cooperatively to stimulate the transcription of their own genes as well as a network of genes required for embryogenesis. Moreover, small changes in the levels of Sox2:Oct-3/4 target genes alter the fate of stem cells. Although positive feedforward and feedback loops have been proposed to explain the activation of these genes, little is known about the mechanisms that prevent their overexpression. Here, we demonstrate that elevating Sox2 levels inhibits the endogenous expression of five Sox2:Oct-3/4 target genes. In addition, we show that Sox2 repression is dependent on the binding sites for Sox2 and Oct-3/4. We also demonstrate that inhibition is dependent on the C-terminus of Sox2, which contains its transactivation domain. Finally, our studies argue that overexpression of neither Oct-3/4 nor Nanog broadly inhibits Sox2:Oct-3/4 target genes. Collectively, these studies provide new insights into the diversity of mechanisms that control Sox2:Oct-3/4 target genes and argue that Sox2 functions as a molecular rheostat for the control of a key transcriptional regulatory network.

Proteomic analysis of Sox2-associated proteins during early stages of mouse embryonic stem cell differentiation identifies Sox21 as a novel regulator of stem cell fate

Small increases in the levels of master regulators, such as Sox2, in embryonic stem cells (ESC) have been shown to promote their differentiation. However, the mechanism by which Sox2 controls the fate of ESC is poorly understood. In this study, we employed multidimensional protein identification technology and identified >60 nuclear proteins that associate with Sox2 early during ESC differentiation. Gene ontology analysis of Sox2‐associated proteins indicates that they participate in a wide range of processes. Equally important, a significant number of the Sox2‐associated proteins identified in this study have been shown previously to interact with Oct4, Nanog, Sall4, and Essrb. Moreover, we examined the impact of manipulating the expression of a Sox2‐associated protein on the fate of ESC. Using ESC engineered for inducible expression of Sox21, we show that ectopic expression of Sox21 in ESC induces their differentiation into specific cell types, including those that express markers representative of neurectoderm and heart development. Collectively, these studies provide new insights into the range of molecular processes through which Sox2 is likely to influence the fate of ESC and provide further support for the conclusion that the expression of Sox proteins in ESC must be precisely regulated. Importantly, our studies also argue that Sox2, along with other pluripotency‐associated transcription factors, is woven into highly interconnected regulatory networks that function at several levels to control the fate of ESC. STEM CELLS 2010;28:1715–1727

Determination of Protein Interactome of Transcription Factor Sox2 in Embryonic Stem Cells Engineered for Inducible Expression of Four Reprogramming Factors

Background: The Sox2-protein interactome in ESC has not been identified. Results: ESC that exogenously express Oct4, Sox2, Klf4, and c-Myc self-renew. This permitted the identification of the Sox2-interactome in ESC. Conclusion: Sox2 associates with >70 proteins, and the knockdown of the Sox2-associated protein Smarcd1 induces the differentiation of ESC. Significance: This is the first description of the Sox2-interactome in undifferentiated ESC. Unbiased proteomic screens provide a powerful tool for defining protein-protein interaction networks. Previous studies employed multidimensional protein identification technology to identify the Sox2-interactome in embryonic stem cells (ESC) undergoing differentiation in response to a small increase in the expression of epitope-tagged Sox2. Thus far the Sox2-interactome in ESC has not been determined. To identify the Sox2-interactome in ESC, we engineered ESC for inducible expression of different combinations of epitope-tagged Sox2 along with Oct4, Klf4, and c-Myc. Epitope-tagged Sox2 was used to circumvent the lack of suitable Sox2 antibodies needed to perform an unbiased proteomic screen of Sox2-associated proteins. Although i-OS-ESC differentiate when both Oct4 and Sox2 are elevated, i-OSKM-ESC do not differentiate even when the levels of the four transcription factors are coordinately elevated ∼2–3-fold. Our findings with i-OS-ESC and i-OSKM-ESC provide new insights into the reasons why ESC undergo differentiation when Sox2 and Oct4 are elevated in ESC. Importantly, the use of i-OSKM-ESC enabled us to identify the Sox2-interactome in undifferentiated ESC. Using multidimensional protein identification technology, we identified >70 proteins that associate with Sox2 in ESC. We extended these findings by testing the function of the Sox2-assoicated protein Smarcd1 and demonstrate that knockdown of Smarcd1 disrupts the self-renewal of ESC and induces their differentiation. Together, our work provides the first description of the Sox2-interactome in ESC and indicates that Sox2 along with other master regulators is part of a highly integrated protein-protein interaction landscape in ESC.

Sox2 Uses Multiple Domains to Associate with Proteins Present in Sox2-Protein Complexes

Master regulators, such as Sox2, Oct4 and Nanog, control complex gene networks necessary for the self-renewal and pluripotency of embryonic stem cells (ESC). These master regulators associate with co-activators and co-repressors to precisely control their gene targets. Recent studies using proteomic analysis have identified a large, diverse group of co-activators and co-repressors that associate with master regulators, including Sox2. In this report, we examined the size distribution of nuclear protein complexes containing Sox2 and its associated proteins HDAC1, Sall4 and Lin28. Interestingly, we determined that Sox2 and HDAC1 associate with protein complexes that vary greatly in size; whereas, Lin28 primarily associates with smaller complexes, and Sall4 primarily associates with larger complexes. Additionally, we examined the domains of Sox2 necessary to mediate its association with its partner proteins Sall4, HDAC1 and HDAC2. We determined that Sox2 uses multiple and distinct domains to associate with its partner proteins. We also examined the domains of Sox2 necessary to mediate its self-association, and we determined that Sox2 self-association is mediated through multiple domains. Collectively, these studies provide novel insights into how Sox2 is able to associate with a wide array of nuclear proteins that control gene transcription.

Musashi2 Is Required for the Self-Renewal and Pluripotency of Embryonic Stem Cells

Recent studies have shown that the RNA binding protein Musashi 2 (Msi2) plays important roles during development. Msi2 has also been shown to be elevated in several leukemias and its elevated expression has been linked with poorer prognosis in these cancers. Additionally, in embryonic stem cells (ESC) undergoing the early stages of differentiation, Msi2 has been shown to associate with the transcription factor Sox2, which is required for the self-renewal of ESC. These findings led us to examine the effects of Msi2 on the behavior of ESC. We determined that ESC express two isoforms of Msi2, the larger canonical isoform (isoform 1) and a shorter, splice-variant isoform (isoform 2). Using multiple shRNA lentiviral vectors, we determined that knockdown of Msi2 disrupts the self-renewal of ESC and promotes their differentiation into cells that express markers associated with mesoderm, ectoderm, and trophectoderm. Moreover, our studies indicate that the extent of differentiation and the loss of self-renewal capacity correlate with the levels to which Msi2 levels were decreased. We extended these findings by engineering ESC to inducibly express either Msi2 isoform1 or isoform 2. We determined that ectopic expression of Msi2 isoform 1, but not isoform 2, enhances the cloning efficiency of ESC. In addition, we examined how Msi2 isoform 1 and isoform 2 affect the differentiation of ESC. Interestingly, ectopic expression of either Msi2 isoform 1 or isoform 2 does not affect the pattern of differentiation induced by retinoic acid. Finally, we show that ectopic expression of either isoform 1 or isoform 2 is not sufficient to block the differentiation that results from the knockdown of both isoforms of Msi2. Thus, it appears that both isoforms of Msi2 are required for the self-renewal of ESC.

Systems Biology Provides New Insights into the Molecular Mechanisms that Control the Fate of Embryonic Stem Cells

During the last 5 years there has been enormous progress in developing a deeper understanding of the molecular mechanisms that control the self‐renewal and pluripotency of embryonic stem cells (ESC). Early progress resulted from studying individual transcription factors and signaling pathways. Unexpectedly, these studies demonstrated that small changes in the levels of master regulators, such as Oct4 and Sox2, promote the differentiation of ESC. More recently, impressive progress has been made using technologies that provide a global view of the signaling pathways and the gene regulatory networks that control the fate of ESC. This review provides an overview of the progress made using several different high‐throughput technologies and focuses on proteomic studies, which provide the first glimpse of the protein–protein interaction networks used by ESC. The latter studies indicate that transcription factors required for the self‐renewal of ESC are part of a large, highly integrated protein–protein interaction landscape, which helps explain why the levels of master regulators need to be regulated precisely in ESC. J. Cell. Physiol. 227: 27–34, 2012.

Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery

The rapid formation of numerous tissues during development is highly dependent on the swift activation of key developmental regulators. Recent studies indicate that many key regulatory genes are repressed in embryonic stem cells (ESCs), yet poised for rapid activation due to the presence of both activating (H3K4 trimethylation) and repressive (H3K27 trimethylation) histone modifications (bivalent genes). However, little is known about bivalent gene regulation. In this study, we investigated the regulation of the bivalent gene Sox21, which is activated rapidly when ESCs differentiate in response to increases in Sox2. Chromatin immunoprecipitation demonstrated that prior to differentiation, the Sox21 gene is bound by a complex array of repressive and activating transcriptional machinery. Upon activation, all identified repressive machinery and histone modifications associated with the gene are lost, but the activating modifications and tran‐scriptional machinery are retained. Notably, these changes do not occur when ESCs differentiate in response to retinoic acid. Moreover, ESCs lacking a functional PRC2 complex fail to activate this gene, apparently due to its association with other repressive complexes. Together, these findings suggest that bivalent genes, such as Sox21, are silenced by a complex set of redundant repressive machinery, which exit rapidly in response to appropriate differentiation signals.—Chakravarthy, H., Ormsbee, B. D., Mallanna, S. K., Rizzino, A. Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene‐silencing machinery. FASEB J. 25, 206–218 (2011). www.fasebj.org

Differential regulation of the Oct‐3/4 gene in cell culture model systems that parallel different stages of mammalian development

Oct‐3/4 is an essential transcription factor that regulates stem cell fate during embryogenesis. Previous reports have shown that the Oct‐3/4 gene utilizes different enhancers to regulate its expression as development proceeds. However, the cis‐elements contributing to the differential activity of these enhancers require further study. Here, we investigated the function of the HMG/POU cassette and LRH‐1 site present in the distal enhancer (DE) and the proximal enhancer, respectively. F9 and P19 EC cells were the focus of this study because their differential utilization of Oct‐3/4 enhancers parallels the use of these enhancers during different stages of development. We determined that the LRH‐1 site functions as a positive and a negative cis‐regulatory element in P19 and F9 EC cells, respectively. Furthermore, we determined that the HMG/POU cassette in the DE strongly activates the Oct‐3/4 promoter in F9 cells, but is a much weaker positive regulatory element in P19 cells. Given that HMG/POU cassettes play key roles in the regulation of at least seven essential genes, the Oct‐3/4 HMG/POU cassette was examined more closely by focusing on Sox2, which can bind to HMG/POU cassettes. Although chromatin immunoprecipitation demonstrated that Sox2 binds to the Oct‐3/4 gene equally well in both EC cell lines, tethering Sox2 to the region of the HMG/POU cassette only activated the Oct‐3/4 promoter in F9 EC cells. These and other findings suggest that the differential activity of the HMG/POU cassette of the Oct‐3/4 gene in EC cells is due to differential action of Sox2 and its associated co‐factors. Mol. Reprod. Dev. 75: 1247–1257, 2008.

Regulation of the Nanog Gene by Both Positive and Negative cis-Regulatory Elements in Embryonal Carcinoma Cells and Embryonic Stem Cells

The transcription factor Nanog is essential for mammalian embryogenesis, as well as the pluripotency of embryonic stem (ES) cells. Work with ES cells and embryonal carcinoma (EC) cells previously identified positive and negative cis‐regulatory elements that influence the activity of the Nanog promoter, including adjacent cis‐regulatory elements that bind Sox2 and Oct‐3/4. Given the importance of Nanog during mammalian development, we examined the cis‐regulatory elements required for Nanog promoter activity more closely. In this study, we demonstrate that two positive cis‐regulatory elements previously shown to be active in F9 EC cells are also active in ES cells. We also identify a novel negative regulatory region that is located in close proximity to two other positive Nanog cis‐regulatory elements. Although this negative regulatory region is active in F9 EC cells and ES cells, it is inactive in P19 EC cells. Furthermore, we demonstrate that one of the positive cis‐regulatory elements active in F9 EC cells and ES cells is inactive in P19 EC cells. Together, these and other studies suggest that Nanog transcription is regulated by the interplay of positive and negative cis‐regulatory elements. Given that P19 appears to be more closely related to a later developmental stage of mammalian development than F9 and ES cells, differential utilization of cis‐regulatory elements may reflect mechanisms used during development to achieve the correct level of Nanog expression as embryogenesis unfolds. Mol. Reprod. Dev. 76: 173–182, 2009.