Kristen L. Kroll

Regulation of Smad signaling through a differential recruitment of coactivators and corepressors by ZEB proteins

Balancing signals derived from the TGFβ family is crucial for regulating cell proliferation and differentiation, and in establishing the embryonic axis during development. TGFβ/BMP signaling leads to the activation and nuclear translocation of Smad proteins, which activate transcription of specific target genes by recruiting P/CAF and p300. The two members of the ZEB family of zinc finger factors (ZEB‐1/δEF1 and ZEB‐2/SIP1) regulate TGFβ/BMP signaling in opposite ways: ZEB‐1/δEF1 synergizes with Smad‐mediated transcriptional activation, while ZEB‐2/SIP1 represses it. Here we report that these antagonistic effects by the ZEB proteins arise from the differential recruitment of transcriptional coactivators (p300 and P/CAF) and corepressors (CtBP) to the Smads. Thus, while ZEB‐1/δEF1 binds to p300 and promotes the formation of a p300–Smad transcriptional complex, ZEB‐2/SIP1 acts as a repressor by recruiting CtBP. This model of regulation by ZEB proteins also functions in vivo, where they have opposing effects on the regulation of TGFβ family‐dependent genes during Xenopus development.

Neurogenin and NeuroD direct transcriptional targets and their regulatory enhancers

Proneural basic helix–loop–helix proteins are key regulators of neurogenesis but their ‘proneural’ function is not well understood, partly because primary targets have not been systematically defined. Here, we identified direct transcriptional targets of the bHLH proteins Neurogenin and NeuroD and found that primary roles of these transcription factors are to induce regulators of transcription, signal transduction, and cytoskeletal rearrangement for neuronal differentiation and migration. We determined targets induced in both Xenopus and mouse, which represent evolutionarily conserved core mediators of Neurogenin and NeuroD activities. We defined consensus sequences for Neurogenin and NeuroD binding and identified responsive enhancers in seven shared target genes. These enhancers commonly contained clustered, conserved consensus‐binding sites and drove neural‐restricted transgene expression in Xenopus embryos. We then used this enhancer signature in a genome‐wide computational approach to predict additional Neurogenin/NeuroD target genes involved in neurogenesis. Taken together, these data demonstrate that Neurogenin and NeuroD preferentially recognize neurogenesis‐related targets through an enhancer signature of clustered consensus‐binding sites and regulate neurogenesis by activating a core set of transcription factors, which build a robust network controlling neurogenesis.

The SWI/SNF chromatin remodeling protein Brg1 is required for vertebrate neurogenesis and mediates transactivation of Ngn and NeuroD

Chromatin remodeling complexes play crucial roles in transcription and are implicated in processes including cell proliferation, differentiation and embryonic patterning. Brg1 is the catalytic subunit of the SWI/SNF chromatin remodeling complex and shows neural-enriched expression. Although early lethality of Brg1-null mice reflects its importance in embryogenesis, this phenotype precluded further study of specific Brg1-dependent developmental processes. Here, we have identified a requirement of Brg1 for both Xenopus primary neurogenesis and neuronal differentiation of mammalian P19 embryonic carcinoma cells. In Xenopus, loss of Brg1 function did not affect neural induction or neural cell fate determination. However, the Sox2-positive, proliferating neural progenitor cell population was expanded, and expression of a terminally differentiated neuronal marker, N-tubulin, was diminished upon loss of Brg1 activity, suggesting that Brg1 is required for neuronal differentiation. The ability of the bHLH transcription factors Ngnr1 and NeuroD to drive neuronal differentiation was also abolished by loss of Brg1 function, indicating that Brg1 is essential for the proneural activities of Ngnr1 and NeuroD. Consistent with this, dominant-negative interference with Brg1 function in P19 cells suppressed neuronal differentiation promoted by NeuroD2, showing the requirement of Brg1 for neuronal differentiation is conserved in mammalian cells. Finally, we discovered that Brg1 physically associates with both Ngnr1 and NeuroD and that interference with Brg1 function blocks Neurogenin3- and NeuroD2-mediated reporter gene transactivation. Together, our results demonstrate that Brg1 (and by inference the SWI/SNF complex) is required for neuronal differentiation by mediating the transcriptional activities of proneural bHLH proteins.

Ajuba LIM proteins are snail/slug corepressors required for neural crest development in Xenopus.

Snail family transcriptional repressors regulate epithelial mesenchymal transitions during physiological and pathological processes. A conserved SNAG repression domain present in all vertebrate Snail proteins is necessary for repressor complex assembly. Here, we identify the Ajuba family of LIM proteins as functional corepressors of the Snail family via an interaction with the SNAG domain. Ajuba LIM proteins interact with Snail in the nucleus on endogenous E-cadherin promoters and contribute to Snail-dependent repression of E-cadherin. Using Xenopus neural crest as a model of in vivo Snail- or Slug-induced EMT, we demonstrate that Ajuba LIM proteins contribute to neural crest development as Snail/Slug corepressors and are required for in vivo Snail/Slug function. Because Ajuba LIM proteins are also components of adherens junctions and contribute to their assembly or stability, their functional interaction with Snail proteins in the nucleus suggests that Ajuba LIM proteins are important regulators of epithelia dynamics communicating surface events with nuclear responses.

Geminin's double life : Chromatin connections that regulate transcription at the transition from proliferation to differentiation

During embryonic development, transitions between cellular programs regulating progenitor cellproliferation and differentiation must be precisely coordinated and temporally controlled toensure that a proper number of cells are allocated to various structures. The novel coiled-coilprotein Geminin was previously characterized as a dual function molecule with roles both inmaintenance of genome integrity through regulation of DNA replication licensing and in controlof neural cell fate during embryonic development. However, the mechanistic basis of Gemininsactivities during embryogenesis and the connections to its cell cycle regulatory role wereunknown. Recently, some of Geminins activities in regulating transcription were shown tooccur through interactions with Brg1, the catalytic subunit of the SWI/SNF chromatinremodeling complex. During development of the nervous system, Geminin controls thetransition from proliferating precursor to differentiated post-mitotic neuron by modulatinginteractions between SWI/SNF and bHLH transcription factors that are critical for neurogenesis.In other developmental contexts, Geminin mediates proliferative-differentiative transitionsthrough interactions with Six3 and Hox transcription factors and Polycomb Group proteins.Interactions of Geminin with Polycomb and SWI/SNF complex proteins link its transcriptionalactivities to modulation of chromatin structure. Here we incorporate recent findings regardingGeminins regulatory roles in coordinating proliferation and differentiation duringembryogenesis.

Neural induction takes a transcriptional twist

Over the past decade, several molecules have been identified that influence neural cell fate in vertebrate embryos during gastrulation. The first neural inducers studied were proteins produced by dorsal mesoderm (the Spemann organizer); most of these proteins act by directly binding to and antagonizing the function of bone morphogenetic proteins (BMPs). Recent experiments have suggested that other secreted signals, such as Wnt and FGF, may neuralize ectoderm before organizer function by a different mechanism. Neural effector genes that mediate the response of ectoderm to secreted neuralizing signals have also been discovered. Interestingly, most of these newly identified neuralizing pathways continue the theme of BMP antagonism, but rather than antagonizing BMP protein function, they may neuralize tissue by suppressing Bmp expression. Down‐regulation of Bmp expression in the prospective neural plate during gastrulation seems to be a shared feature of neural induction in vertebrate embryos. However, the signals used to accomplish this task seem to vary among vertebrates. Here, we will discuss the role of the recently identified secreted signals and neural effector genes in vertebrate neurogenesis.

Geminin promotes neural fate acquisition of embryonic stem cells by maintaining chromatin in an accessible and hyperacetylated state

Formation of the complex vertebrate nervous system begins when pluripotent cells of the early embryo are directed to acquire a neural fate. Although cell intrinsic controls play an important role in this process, the molecular nature of this regulation is not well defined. Here we assessed the role for Geminin, a nuclear protein expressed in embryonic cells, during neural fate acquisition from mouse embryonic stem (ES) cells. Whereas Geminin knockdown does not affect the ability of ES cells to maintain or exit pluripotency, we found that it significantly impairs their ability to acquire a neural fate. Conversely, Geminin overexpression promotes neural gene expression, even in the presence of growth factor signaling that antagonizes neural transcriptional responses. These data demonstrate that Geminins activity contributes to mammalian neural cell fate acquisition. We investigated the mechanistic basis of this phenomenon and found that Geminin maintains a hyperacetylated and open chromatin conformation at neural genes. Interestingly, recombinant Geminin protein also rapidly alters chromatin acetylation and accessibility even when Geminin is combined with nuclear extract and chromatin in vitro. Together, these data support a role for Geminin as a cell intrinsic regulator of neural fate acquisition that promotes expression of neural genes by regulating chromatin accessibility and histone acetylation.

Geminin cooperates with Polycomb to restrain multi-lineage commitment in the early embryo

Transient maintenance of a pluripotent embryonic cell population followed by the onset of multi-lineage commitment is a fundamental aspect of development. However, molecular regulation of this transition is not well characterized in vivo. Here, we demonstrate that the nuclear protein Geminin is required to restrain commitment and spatially restrict mesoderm, endoderm and non-neural ectoderm to their proper locations in the Xenopus embryo. We used microarray analyses to demonstrate that Geminin overexpression represses many genes associated with cell commitment and differentiation, while elevating expression levels of genes that maintain pluripotent early and immature neurectodermal cell states. We characterized the relationship of Geminin to cell signaling and found that Geminin broadly represses Activin-, FGF- and BMP-mediated cell commitment. Conversely, Geminin knockdown enhances commitment responses to growth factor signaling and causes ectopic mesodermal, endodermal and epidermal fate commitment in the embryo. We also characterized the functional relationship of Geminin with transcription factors that had similar activities and found that Geminin represses commitment independent of Oct4 ortholog (Oct25/60) activities, but depends upon intact Polycomb repressor function. Consistent with this, chromatin immunoprecipitation assays directed at mesodermal genes demonstrate that Geminin promotes Polycomb binding and Polycomb-mediated repressive histone modifications, while inhibiting modifications associated with gene activation. This work defines Geminin as an essential regulator of the embryonic transition from pluripotency through early multi-lineage commitment, and demonstrates that functional cooperativity between Geminin and Polycomb contributes to this process.

Geminin in embryonic development: coordinating transcription and the cell cycle during differentiation.

Geminin was initially characterized as a bifunctional protein with roles in regulating the fidelity of DNA replication and in controlling cell fate during embryonic nervous system formation. More recently, Geminins roles have expanded, encompassing regulation of cell proliferation and differentiation during retinogenesis, control of Hox transcription factor function during vertebrate axial patterning, and regulation of the timing of neuronal differentiation. Geminin interacts with homeodomain-containing transcription factors and with protein complexes that regulate chromatin structure, including Polycomb complexes and the catalytic subunits of the SWI/SNF chromatin remodeling complex, Brg1 and Brahma. Activities for Geminin in coordinating cellular events at the transition from proliferation to differentiation have recently emerged in multiple developmental contexts. This review will summarize Geminins increasingly diverse roles as a developmental regulatory molecule.

A Phosphomimetic Mutation in the Sall1 Repression Motif Disrupts Recruitment of the Nucleosome Remodeling and Deacetylase Complex and Repression of Gbx2

The multizinc finger transcription factor Sall1 is a critical developmental regulator that mediates repression through the recruitment of the nucleosome remodeling and deacetylase (NuRD) complex. Although a short conserved peptide motif in Sall1 is sufficient to recruit NuRD, its ability to regulate native Sall1 target genes in vivo has not been demonstrated. In this report, we demonstrate an in vivo role for the Sall1 repression motif and describe a novel direct target gene of Sall1, Gbx2, that is directly repressed in a NuRD-dependent fashion. The ability of Sall1 to repress Gbx2 was impaired in Xenopus embryos expressing mutant forms of Sall1 that are defective for NuRD binding. Finally, we demonstrate that protein kinase C phosphorylates serine 2 of the Sall1 repression motif and reveal that a phosphomimetic mutation of serine 2 disrupts the ability of Sall1 to repress Gbx2 in cell culture and Xenopus embryos. Together, these studies establish that Sall1 recruits NuRD via the Sall1 repression motif to mediate repression of a native target gene and suggest a model in which dynamic control of gene expression by Sall1 is modulated by serine phosphorylation of the Sall1 repression motif.