Cell Fate-determining Transcription Factors Research Articles

HMX3 is a critical vulnerability in MECOM-negative KMT2A::MLLT3 acute myelomonocytic leukemia.

KMT2A::MLLT3 acute myelomonocytic leukemia (AML) comes in two clinically and biologically different subtypes. One is characterized by inferior outcome, older age, and MECOM oncogene expression. The other is mainly observed in children and young adults, associates with better clinical outcome, but lacks MECOM. To identify cell fate determining transcription factors downstream of KMT2A::MLLT3, we applied a bioinformatic algorithm that integrates gene and enhancer expression from primary MECOM-positive and -negative KMT2A::MLLT3 AML samples. This identified MECOM to be most influential in the MECOM-positive group, while neuronal transcription factor HMX3 was most influential in the MECOM-negative group. In large AML cohorts, HMX3 expression associated with a unique gene expression profile, younger age (p < 0.002) and KMT2A-rearranged and KAT6A-CREBBP leukemia (p < 0.00001). HMX3 was not expressed in other major genetic risk groups and healthy blood cells. RNA-sequencing analyses following forced HMX3 expression in healthy CD34+ cells and its silencing in KMT2A::MLT3 cells showed that HMX3 drives cancer-associated E2F and MYC gene programs (p < 0.001). HMX3 expression in healthy CD34+ cells blocked monocytic but not granulocytic colony formation. Strikingly, HMX3 silencing in KMT2A::MLLT3 patient cells resulted in cell cycle arrest, monocytic differentiation and apoptosis. Thus, the neuronal transcription factor HMX3 is a leukemia-specific vulnerability in KMT2A::MLLT3 AML.

Generation of a stably transfected mouse embryonic stem cell line for inducible differentiation to excitatory neurons

In vitro differentiation of stem cells into various cell lineages is valuable in developmental studies and an important source of cells for modelling physiology and pathology, particularly for complex tissues such as the brain. Conventional protocols for in vitro neuronal differentiation often suffer from complicated procedures, high variability and low reproducibility. Over the last decade, the identification of cell fate-determining transcription factors has provided new tools for cellular studies in neuroscience and enabled rapid differentiation driven by ectopic transcription factor expression. As a proneural transcription factor, Neurogenin 2 (Ngn2) expression alone is sufficient to trigger rapid and robust neurogenesis from pluripotent cells. Here, we established a stable cell line, by piggyBac (PB) transposition, that conditionally expresses Ngn2 for generation of excitatory neurons from mouse embryonic stem cells (ESCs) using an all-in-one PB construct. Our results indicate that Ngn2-induced excitatory neurons have mature and functional characteristics consistent with previous studies using conventional differentiation methods. This approach provides an all-in-one PB construct for rapid and high copy number gene delivery of dox-inducible transcription factors to induce differentiation. This approach is a valuable in vitro cell model for disease modeling, drug screening and cell therapy.

Vitamin D receptor cross-talk with p63 signaling promotes epidermal cell fate

The vitamin D receptor with its ligand 1,25 dihydroxy vitamin D3 (1,25D3) regulates epidermal stem cell fate, such that VDR removal from Krt14 expressing keratinocytes delays re-epithelialization of epidermis after wound injury in mice. In this study we deleted Vdr from Lrig1 expressing stem cells in the isthmus of the hair follicle then used lineage tracing to evaluate the impact on re-epithelialization following injury. We showed that Vdr deletion from these cells prevents their migration to and regeneration of the interfollicular epidermis without impairing their ability to repopulate the sebaceous gland. To pursue the molecular basis for these effects of VDR, we performed genome wide transcriptional analysis of keratinocytes from Vdr cKO and control littermate mice. Ingenuity Pathway analysis (IPA) pointed us to the TP53 family including p63 as a partner with VDR, a transcriptional factor that is essential for proliferation and differentiation of epidermal keratinocytes. Epigenetic studies on epidermal keratinocytes derived from interfollicular epidermis showed that VDR is colocalized with p63 within the specific regulatory region of MED1 containing super-enhancers of epidermal fate driven transcription factor genes such as Fos and Jun. Gene ontology analysis further implicated that Vdr and p63 associated genomic regions regulate genes involving stem cell fate and epidermal differentiation. To demonstrate the functional interaction between VDR and p63, we evaluated the response to 1,25(OH)2D3 of keratinocytes lacking p63 and noted a reduction in epidermal cell fate determining transcription factors such as Fos, Jun. We conclude that VDR is required for the epidermal stem cell fate orientation towards interfollicular epidermis. We propose that this role of VDR involves cross-talk with the epidermal master regulator p63 through super-enhancer mediated epigenetic dynamics.

Transcriptional programming and gene regulation in WC1+ γδ T cell subpopulations

γδ T cells represent a high proportion of lymphocytes in the blood of ruminants with the majority expressing lineage-specific glycoproteins from the WC1 family. WC1 receptors are coded for by a multigenic array whose genes have variegated but stable expression among cells in the γδ T cell population. WC1 molecules function as hybrid pattern recognition receptors as well as co-receptors for the TCR and are required for responses by the cells. Because of the variegated gene expression, WC1+ γδ T cells can be divided into two main populations known as WC1.1+ and WC1.2+ based on monoclonal antibody reactivity with the expressed WC1 molecules. These subpopulations differ in their ability to respond to specific pathogens. Here, we showed these populations are established in the thymus and that WC1.1+ and WC1.2+ subpopulations have transcriptional programming that is consistent with stratification towards Tγδ1 or Tγδ17. WC1.1+ cells exhibited the Tγδ1 phenotype with greater transcription of Tbx21 and production of more IFNγ while the WC1.2+ subpopulation tended towards Tγδ17 programming producing higher levels of IL-17 and had greater transcription of Rorc. However, when activated both WC1+ subpopulations' cells transcribed Tbx21 and secreted IFNγ and IL-17 reflecting the complexity of these subpopulations defined by WC1 gene expression. The gene networks involved in development of these two subpopulations including expression of their archetypal genes wc1-3 (WC1.1+) and wc1-4 (WC1.2+) were unknown but we report that SOX-13, a γδ T cell fate-determining transcription factor, has differential occupancy on these WC1 gene loci and suggest a model for development of these subpopulations.

NGN2 mmRNA-Based Transcriptional Programming in Microfluidic Guides hiPSCs Toward Neural Fate With Multiple Identities.

Recent advancements in cell engineering have succeeded in manipulating cell identity with the targeted overexpression of specific cell fate determining transcription factors in a process named transcriptional programming. Neurogenin2 (NGN2) is sufficient to instruct pluripotent stem cells (PSCs) to acquire a neuronal identity when delivered with an integrating system, which arises some safety concerns for clinical applications. A non-integrating system based on modified messenger RNA (mmRNA) delivery method, represents a valuable alternative to lentiviral-based approaches. The ability of NGN2 mmRNA to instruct PSC fate change has not been thoroughly investigated yet. Here we aimed at understanding whether the use of an NGN2 mmRNA-based approach combined with a miniaturized system, which allows a higher transfection efficiency in a cost-effective system, is able to drive human induced PSCs (hiPSCs) toward the neuronal lineage. We show that NGN2 mRNA alone is able to induce cell fate conversion. Surprisingly, the outcome cell population accounts for multiple phenotypes along the neural development trajectory. We found that this mixed population is mainly constituted by neural stem cells (45% ± 18 PAX6 positive cells) and neurons (38% ± 8 βIIITUBULIN positive cells) only when NGN2 is delivered as mmRNA. On the other hand, when the delivery system is lentiviral-based, both providing a constant expression of NGN2 or only a transient pulse, the outcome differentiated population is formed by a clear majority of neurons (88% ± 1 βIIITUBULIN positive cells). Altogether, our data confirm the ability of NGN2 to induce neuralization in hiPSCs and opens a new point of view in respect to the delivery system method when it comes to transcriptional programming applications.

Transcription Factor-Based Fate Specification and Forward Programming for Neural Regeneration.

Traditionally, in vitro generation of donor cells for brain repair has been dominated by the application of extrinsic growth factors and morphogens. Recent advances in cell engineering strategies such as reprogramming of somatic cells into induced pluripotent stem cells and direct cell fate conversion have impressively demonstrated the feasibility to manipulate cell identities by the overexpression of cell fate-determining transcription factors. These strategies are now increasingly implemented for transcription factor-guided differentiation of neural precursors and forward programming of pluripotent stem cells toward specific neural subtypes. This review covers major achievements, pros and cons, as well as future prospects of transcription factor-based cell fate specification and the applicability of these approaches for the generation of donor cells for brain repair.

IA26 Stage-Specific Roles of RB Constrain Tumor Progression, Lineage Fidelity, and Metastasis

Mutations in the Rb tumor suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma. Additionally, recent clinical successes with cyclin-dependent kinase inhibitors have reinvigorated interest in reactivating the Retinoblastoma (Rb) pathway to treat lung adenocarcinoma and other tumor types. Remarkably, though, Rb’s role in suppressing lung adenocarcinoma remains unclear and whether Rb pathway reactivation would be efficacious in this disease remains unknown. To model Rb pathway reactivation as a treatment strategy in lung adenocarcinoma and to shed light on its role in this disease, we established an RbXTR allele that enables Cre-dependent inactivation of Rb in developing tumors and allows Flp recombinase-inducible reactivation of Rb after tumors are established. In the KrasLox-Stop-Lox-G12D/+;p53flox/flox (KP) mouse model of lung adenocarcinoma, we show that Rb inactivation facilitates the bypass of two molecularly distinct barriers to tumor progression and dramatically accelerates malignant conversion and the development of metastatic disease. Although in the presence of Rb, malignant conversion requires amplification of the Raf/Mek/Erk (MAPK) signaling pathway beyond that normally activated by the Kras oncogene, we find that this requirement is abrogated when Rb is inactivated. Mechanistically, we identified Cdk2 as an important effector downstream of amplified MAPK signaling and that this activity suppresses Rb’s ability to limit the adenoma-to-carcinoma transition. Importantly, inactivation of Cdk2 reduces cell proliferation in Rb wild-type cells and confers sensitivity to Cdk4/6 inhibition in both human and mouse lung adenocarcinoma cell lines that were intrinsically resistant. Acquiring metastatic competency in Rb wild-type tumors is causally linked to epigenetic changes resulting in loss of lung lineage cell fate-determining transcription factors and concomitant derepression of factors normally restricted to embryonic cell types. However, inactivation of Rb uncouples the onset of metastatic competency from the loss of lung lineage factors, facilitates the early derepression of prometastatic factors, and significantly enhances metastatic proclivity. Finally, we demonstrate that reactivation of Rb in metastatic disease settings reprograms these tumors toward a less aggressive cell state and improves overall survival. Our study highlights an unappreciated role for Rb in regulating metastasis-promoting programs, and the potential of Rb restorative therapies to treat lung adenocarcinoma. Further, we suggest a renewed investment in the development of specific Cdk2 inhibitors may be necessary for Rb pathway reactivation in certain cancer types.

Selective sequestration of signalling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus.

Selective recruitment and concentration of signaling proteins within membraneless compartments is a ubiquitous mechanism for subcellular organization1–3. The dynamic flow of molecules into and out of these compartments occurs on faster timescales than for membrane-enclosed organelles, presenting a possible mechanism to control spatial patterning within cells. Here, we combined single-molecule tracking and super-resolution microscopy, light-induced subcellular localization, reaction-diffusion modeling, and a spatially-resolved promoter activation assay to study signal exchange in and out of the 200 nm cytoplasmic PopZ microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus4–8. Two phospho-signaling proteins, the transmembrane histidine kinase CckA and the cytoplasmic phosphotransferase ChpT, provide the only phosphate source for the cell fate-determining transcription factor CtrA (Fig. 1a)9–18. We found that all three proteins exhibit restricted rates of entry into and escape from the microdomain and enhanced phospho-signaling within, leading to a submicron gradient of activated CtrA~P19 that is stable and sublinear. Entry into the microdomain is selective for cytosolic proteins and requires a binding pathway to PopZ. Our work demonstrates how nanoscale protein assemblies can modulate signal propagation with fine spatial-resolution, and that in Caulobacter, this modulation serves to reinforce asymmetry and differential cell fate of the two daughter cells.

Abstract PR01: Modeling Rb loss and pathway reactivation in lung adenocarcinoma

Abstract Mutations in the Rb tumor suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma. Additionally, recent clinical successes with cyclin dependent kinase inhibitors have reinvigorated interest in reactivating the Retinoblastoma (Rb) pathway to treat lung adenocarcinoma and other tumor types. Remarkably though, Rb’s role in suppressing lung adenocarcinoma remains unclear, and whether Rb pathway reactivation would be efficacious in this disease remains unknown. To model Rb pathway reactivation as treatment strategy in lung adenocarcinoma and to shed light on its role in this disease, we established an RbXTR allele that enables Cre-dependent inactivation of Rb in developing tumors, and allows Flp recombinase-inducible reactivation of Rb after tumors are established. In the KrasLox-Stop-Lox-G12D/+;p53flox/flox (KP) mouse model of lung adenocarcinoma, we show that Rb inactivation facilitates the bypass of two molecularly distinct barriers to tumor progression and dramatically accelerates malignant conversion and the development of metastatic disease. Although, in the presence of Rb, malignant conversion requires amplification of the Raf/Mek/Erk (MAPK) signaling pathway beyond that normally activated by the Kras oncogene, we find that this requirement is abrogated when Rb is inactivated. Mechanistically, we identified Cdk2 as an important effector downstream of amplified MAPK signaling and that this activity suppresses Rb’s ability to limit the adenoma-to-carcinoma transition. Importantly, inactivation of Cdk2 reduces cell proliferation in Rb wild-type cells and confers sensitivity to Cdk4/6 inhibition in both human and mouse lung adenocarcinoma cell lines were intrinsically resistant. Acquiring metastatic competency in Rb wild-type tumors is causally linked to epigenetic changes resulting in loss of lung lineage cell fate-determining transcription factors and concomitant derepression of factors normally restricted to embryonic cell types. However, inactivation of Rb uncouples the onset of metastatic competency from the loss of lung lineage factors, facilitates the early derepression of prometastatic factors, and significantly enhances metastatic proclivity. Finally, we demonstrate that reactivation of Rb in metastatic disease settings reprograms these tumors toward a less aggressive cell state and improves overall survival. Our study highlights an unappreciated role for Rb in regulating metastasis-promoting programs, and the potential of Rb restorative therapies to treat lung adenocarcinoma. Further, we suggest that a renewed investment in the development of specific Cdk2 inhibitors may be necessary for Rb pathway reactivation in certain cancer types. This abstract is also being presented as Poster A27. Citation Format: Travis Yates, Caroline Kim-Kiselak, Walter Wang, David M. Feldser. Modeling Rb loss and pathway reactivation in lung adenocarcinoma [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR01.

Abstract 3189: Neuronal transcription factor BRN2 is an androgen suppressed driver of neuroendocrine differentiation in prostate cancer

Abstract Neuroendocrine prostate cancer (NEPC) is an incurable, rapidly progressing and lethal disease. NEPC is increasingly recognized as a highly therapy resistant tumor variant that evolves from castration-resistant prostate cancer (CRPC) in a subset of patients treated with potent androgen receptor (AR) pathway inhibitors like enzalutamide (ENZ). Importantly, mechanisms by which the AR directly controls the emergence of NEPC from CRPC under the selective pressure of ENZ remain elusive. As the AR is the cornerstone therapeutic target in men with CRPC, understanding its contribution to the development of NEPC is critical to better implement current standard-of-care therapies such as ENZ, and to identify novel therapeutic targets for this incurable disease. Hallmarks of NEPC are resistance to ENZ and the loss or reduced activity of the AR. Thus, we hypothesized that a consequence of ENZ treatment and resistance in CRPC is relief of AR-mediated suppression of factors that drive NEPC. To investigate this, we developed a model of prostate cancer that mimics clinical progression to CRPC and ENZ resistance (ENZR CPRC). Mirroring what is observed in some patients who progress on ENZ, 25% of our ENZR CRPC tumors and derived cell lines showed strong reduction in classic activity of the AR and a NEPC phenotype. By interrogating NEPC-like ENZR CRPC and ENZ-treated CRPC tumors and cell lines with RNA-Seq and mechanistic in vitro and in vivo studies, as well as human tumors with RNA-Seq and IHC, we identified the master neural transcription factor BRN2 as a central and clinically relevant driver of NEPC differentiation. Specifically, we found AR binding in the BRN2 enhancer directly represses BRN2 transcription and this release drives NEPC marker expression and aggressive growth of ENZR CRPC. Our data also reveal a striking overlap of AR and SOX binding motifs in the enhancer region of BRN2 creating a competitive binding scenario between AR and SOX2, another cell-fate determining transcription factor associated with NEPC. We discovered that upregulation of BRN2 further enhances SOX2 expression and that a BRN2-SOX2 interaction contributes to NEPC differentiation. Importantly, we found BRN2 is highly expressed in human NEPC and metastatic CRPC, especially in patients with low AR activity, highlighting its clinical relevance to disease that is difficult to treat with mainstay therapies. These data underscore the consequences of potent AR inhibition in CRPC, revealing a novel mechanism of AR-dependent control of NEPC via direct suppression of BRN2. This work uncovers BRN2 as a clinically relevant potential therapeutic target to prevent emergence of NEPC from ENZR CRPC. Citation Format: Jennifer L. Bishop, Daksh Thaper, Sepideh Vahid, Ravi S. Munuganti, Paul Ahn, Alastair Davies, Kirsi Ketola, Ka Mun Nip, Dong Lin, Yuzhuo Wang, Himisha Beltran, Amina Zoubeidi. Neuronal transcription factor BRN2 is an androgen suppressed driver of neuroendocrine differentiation in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3189. doi:10.1158/1538-7445.AM2017-3189

Next-generation-sequencing of recurrent childhood high hyperdiploid acute lymphoblastic leukemia reveals mutations typically associated with high risk patients

20% of children suffering from high hyperdiploid acute lymphoblastic leukemia develop recurrent disease. The molecular mechanisms are largely unknown. Here, we analyzed the genetic landscape of five patients at relapse, who developed recurrent disease without prior high-risk indication using whole-exome- and whole-genome-sequencing. Oncogenic mutations of RAS pathway genes (NRAS, KRAS, FLT3, n=4) and deactivating mutations of major epigenetic regulators (CREBBP, EP300, each n=2 and ARID4B, EZH2, MACROD2, MLL2, each n=1) were prominent in these cases and virtually absent in non-recurrent cases (n=6) or other pediatric acute lymphoblastic leukemia cases (n=18).In relapse nucleotide variations were detected in cell fate determining transcription factors (GLIS1, AKNA). Structural genomic alterations affected genes regulating B-cell development (IKZF1, PBX1, RUNX1). Eleven novel translocations involved the genes ART4, C12orf60, MACROD2, TBL1XR1, LRRN4, KIAA1467, and ELMO1/MIR1200. Typically, patients harbored only single structural variations, except for one patient who displayed massive rearrangements in the context of a germline tumor suppressor TP53 mutation and a Li-Fraumeni syndrome-like family history. Another patient harbored a germline mutation in the DNA repair factor ATM. In summary, the relapse patients of our cohort were characterized by somatic mutations affecting the RAS pathway, epigenetic and developmental programs and germline mutations in DNA repair pathways.

Hybrid T-helper cells: stabilizing the moderate center in a polarized system.

Polarization of cell phenotypes, a common strategy to achieve cell type diversity in metazoa, results from binary cell-fate decisions in the branching pedigree of development. Such “either-or” fate decisions are controlled by two opposing cell fate-determining transcription factors. Each of the two distinct “master regulators” promotes differentiation of its respective sister lineage. But they also suppress one other, leading to their mutually exclusive expression in the two ensuing lineages. Thus, promiscuous coexistence of the antagonist regulators in the same cell, the hallmark of the common “undecided” progenitor of two sister lineages, is considered unstable. This antagonism ensures robust polarization into two discretely distinct cell types. But now the immune system's T-helper (Th) cells and their two canonical subtypes, Th1 and Th2 cells, tell a different story, as revealed in three papers recently published in PLOS Biology. The intermediate state that co-expresses the two opposing master regulators of the Th1 and Th2 subtypes, T-bet and Gata3, is highly stable and is not necessarily an undecided precursor. Instead, the Th1/Th2 hybrid cell is a robust new type with properties of both Th1 and Th2 cells. These hybrid cells are functionally active and possess the benefit of moderation: self-limitation of effector T cell function to prevent excessive inflammation, a permanent risk in host defense that can cause tissue damage or autoimmunity. Gene regulatory network analysis suggests that stabilization of the intermediate center in a polarizing system can be achieved by minor tweaking of the architecture of the mutual suppression gene circuit, and thus is a design option readily available to evolution.

The DNA methylation landscape of small cell lung cancer suggests a differentiation defect of neuroendocrine cells

Small cell lung cancer (SCLC) is a disease characterized by aggressive clinical behavior and lack of effective therapy. Owing to its tendency for early dissemination, only a third of patients have limited-stage disease at the time of diagnosis. SCLC is thought to derive from pulmonary neuroendocrine cells. Although several molecular abnormalities in SCLC have been described, there are relatively few studies on epigenetic alterations in this type of tumor. Here, we have used methylation profiling with the methylated-CpG island recovery assay in combination with microarrays and conducted the first genome-scale analysis of methylation changes that occur in primary SCLC and SCLC cell lines. Among the hundreds of tumor-specifically methylated genes discovered, we identified 73 gene targets that are methylated in >77% of primary SCLC tumors, most of which have never been linked to aberrant methylation in tumors. These methylated targets have potential for biomarker development for early detection and therapeutic management of SCLC. SCLC cell lines had a greater number of hypermethylated genes than primary tumors. Gene ontology analysis indicated a significant enrichment of methylated genes functioning as transcription factors and in processes of neuronal differentiation. Motif analysis of tumor-specific methylated regions identified enrichment of binding sites for several neural cell fate-specifying transcription factors including NEUROD1, HAND1, ZNF423 and REST. We hypothesize that two potential mechanisms, loss of cell fate-determining transcription factors by methylation of their promoters and functional inactivation of their corresponding genomic-binding sites by DNA methylation, can promote a differentiation defect of neuroendocrine cells thus enhancing the ability of tumor progenitor cells to transition toward SCLC.

Epigenetic Gene Silencing by the SRY Protein Is Mediated by a KRAB-O Protein That Recruits the KAP1 Co-repressor Machinery

The sex determination transcription factor SRY is a cell fate-determining transcription factor that mediates testis differentiation during embryogenesis. It may function by repressing the ovarian determinant gene, RSPO1, action in the ovarian developmental pathway and activates genes, such as SOX9, important for testis differentiation at the onset of gonadogenesis. Further, altered expression of SRY and related SOX genes contribute to oncogenesis in many human cancers. Little is known of the mechanisms by which SRY regulates its target genes. Recently a KRAB domain protein (KRAB-O) that lacks a zinc finger motif has been demonstrated to interact with SRY and hypothesized to function as an adaptor molecule for SRY by tethering the KAP1-NuRD-SETDB1-HP1 silencing machinery to repress SRY targets. We have critically examined this hypothesis by reconstituting and characterizing SRY-KRAB-O-KAP1 interactions. These recombinant molecules can form a ternary complex by direct and high affinity interactions. The KRAB-O protein can simultaneously bind KAP1 and SRY in a noncompetitive but also noncooperative manner. An extensive mutagenesis analysis suggests that different surfaces on KRAB-O are utilized for these independent interactions. Transcriptional repression by SRY requires binding to KRAB-O, thus bridging to the KAP1 repression machinery. This repression machinery is recruited to SRY target promoters in chromatin templates via SRY. These results suggest that SRY has co-opted the KRAB-O protein to recruit the KAP1 repression machinery to sex determination target genes. Other KRAB domain proteins, which lack a zinc finger DNA-binding motif, may function in similar roles as adaptor proteins for epigenetic gene silencing.

Sox9 Inhibits Wnt Signaling by Promoting β-Catenin Phosphorylation in the Nucleus

Chondrocyte fate determination and maintenance requires Sox9, an intrinsic transcription factor, but is inhibited by Wnt/beta-catenin signaling activated by extrinsic Wnt ligands. Here we explored the underlying molecular mechanism by which Sox9 antagonizes the Wnt/beta-catenin signaling in chondrocyte differentiation. We found that Sox9 employed two distinct mechanisms to inhibit Wnt/beta-catenin signaling: the Sox9 N terminus is necessary and sufficient to promote beta-catenin degradation, whereas the C terminus is required to inhibit beta-catenin transcriptional activity without affecting its stability. Sox9 binds to beta-catenin and components of the beta-catenin "destruction complex," glycogen synthase kinase 3 and beta-transducin repeat containing protein, to promote their nuclear localization. Independent of its DNA binding ability, nuclear localization of Sox9 is both necessary and sufficient to enhance beta-catenin phosphorylation and its subsequent degradation. Thus, one mechanism whereby Sox9 regulates chondrogenesis is to promote efficient beta-catenin phosphorylation in the nucleus. This mechanism may be broadly employed by other intrinsic cell fate determining transcription factors to promptly turn off extrinsic inhibitory Wnt signaling mediated by beta-catenin.

Mitotic retention of gene expression patterns by the cell fate-determining transcription factor Runx2

During cell division, cessation of transcription is coupled with mitotic chromosome condensation. A fundamental biological question is how gene expression patterns are retained during mitosis to ensure the phenotype of progeny cells. We suggest that cell fate-determining transcription factors provide an epigenetic mechanism for the retention of gene expression patterns during cell division. Runx proteins are lineage-specific transcription factors that are essential for hematopoietic, neuronal, gastrointestinal, and osteogenic cell fates. Here we show that Runx2 protein is stable during cell division and remains associated with chromosomes during mitosis through sequence-specific DNA binding. Using siRNA-mediated silencing, mitotic cell synchronization, and expression profiling, we identify Runx2-regulated genes that are modulated postmitotically. Novel target genes involved in cell growth and differentiation were validated by chromatin immunoprecipitation. Importantly, we find that during mitosis, when transcription is shut down, Runx2 selectively occupies target gene promoters, and Runx2 deficiency alters mitotic histone modifications. We conclude that Runx proteins have an active role in retaining phenotype during cell division to support lineage-specific control of gene expression in progeny cells.

The mechanism of bacterial asymmetric cell division.

Asymmetric cell division generates two cells that contain different regulatory proteins and express different fates. In an example of asymmetric cell division from B. subtilis, a site on the membrane of the dividing cell is chosen to establish the initial asymmetry. Recent results show that a key regulatory protein, SpoIIE, is localized to one side of a sporulating B. subtilis cell, and subsequently functions in an asymmetric manner. SpoIIE is a phosphatase at the beginning of a regulatory cascade that leads to activation of a cell fate-determining transcription factor in only one daughter cell.