Postnatal Conditional Deletion of Bcl11b in Striatal Projection Neurons Mimics the Transcriptional Signature of Huntington's Disease.

The dysregulation of striatal gene expression and function is linked to multiple diseases, including Huntington's disease (HD), Parkinson's disease, X-linked dystonia-parkinsonism (XDP), addiction, autism, and schizophrenia. Striatal medium spiny neurons (MSNs) make up 90% of the neurons in the striatum and are critical to motor control. The transcription factor, Bcl11b (also known as Ctip2), is required for striatal development, but the function of Bcl11b in adult MSNs in vivo has not been investigated. We conditionally deleted Bcl11b specifically in postnatal MSNs and performed a transcriptomic and behavioral analysis on these mice. Multiple enrichment analyses showed that the D9-Cre-Bcl11btm1.1Leid transcriptional profile was similar to the HD gene expression in mouse and human data sets. A Gene Ontology enrichment analysis linked D9-Cre-Bcl11btm1.1Leid to calcium, synapse organization, specifically including the dopaminergic synapse, protein dephosphorylation, and HDAC-signaling, commonly dysregulated pathways in HD. D9-Cre-Bcl11btm1.1Leid mice had decreased DARPP-32/Ppp1r1b in MSNs and behavioral deficits, demonstrating the dysregulation of a subtype of the dopamine D2 receptor expressing MSNs. Finally, in human HD isogenic MSNs, the mislocalization of BCL11B into nuclear aggregates points to a mechanism for BCL11B loss of function in HD. Our results suggest that BCL11B is important for the function and maintenance of mature MSNs and Bcl11b loss of function drives, in part, the transcriptomic and functional changes in HD.

Realization of the T Lineage Program Involves GATA-3 Induction of Bcl11b and Repression of Cdkn2b Expression.

The zinc-finger transcription factor GATA-3 plays a crucial role during early T cell development and also dictates later T cell differentiation outcomes. However, its role and collaboration with the Notch signaling pathway in the induction of T lineage specification and commitment have not been fully elucidated. We show that GATA-3 deficiency in mouse hematopoietic progenitors results in an early block in T cell development despite the presence of Notch signals, with a failure to upregulate Bcl11b expression, leading to a diversion along a myeloid, but not a B cell, lineage fate. GATA-3 deficiency in the presence of Notch signaling results in the apoptosis of early T lineage cells, as seen with inhibition of CDK4/6 (cyclin-dependent kinases 4 and 6) function, and dysregulated cyclin-dependent kinase inhibitor 2b (Cdkn2b) expression. We also show that GATA-3 induces Bcl11b, and together with Bcl11b represses Cdkn2b expression; however, loss of Cdkn2b failed to rescue the developmental block of GATA-3-deficient T cell progenitor. Our findings provide a signaling and transcriptional network by which the T lineage program in response to Notch signals is realized.

Discovery and Validation of a Compound to Target Ewing's Sarcoma.

Ewing's sarcoma, characterized by pathognomonic t (11; 22) (q24; q12) and related chromosomal ETS family translocations, is a rare aggressive cancer of bone and soft tissue. Current protocols that include cytotoxic chemotherapeutic agents effectively treat localized disease; however, these aggressive therapies may result in treatment-related morbidities including second-site cancers in survivors. Moreover, the five-year survival rate in patients with relapsed, recurrent, or metastatic disease is less than 30%, despite intensive therapy with these cytotoxic agents. By using high-throughput phenotypic screening of small molecule libraries, we identified a previously uncharacterized compound (ML111) that inhibited in vitro proliferation of six established Ewing's sarcoma cell lines with nanomolar potency. Proteomic studies show that ML111 treatment induced prometaphase arrest followed by rapid caspase-dependent apoptotic cell death in Ewing's sarcoma cell lines. ML111, delivered via methoxypoly(ethylene glycol)-polycaprolactone copolymer nanoparticles, induced dose-dependent inhibition of Ewing's sarcoma tumor growth in a murine xenograft model and invoked prometaphase arrest in vivo, consistent with in vitro data. These results suggest that ML111 represents a promising new drug lead for further preclinical studies and is a potential clinical development for the treatment of Ewing's sarcoma.

A targeted combinatorial therapy for Ewing's sarcoma.

Ewing's sarcoma (EwS) is the second most common bone cancer in children and adolescents. Current chemotherapy regimens are mainly ineffective in patients with relapsed disease and cause long-term effects in survivors. Therefore, we have developed a combinatorial therapy based on a novel drug candidate named ML111 that exhibits selective activity against EwS cells and synergizes with vincristine. To increase the aqueous solubility of hydrophobic ML111, polymeric nanoparticles (ML111-NP) were developed. In vitro data revealed that ML111-NP compromise viability of EwS cells without affecting non-malignant cells. Furthermore, ML111-NP exhibit strong synergistic effects in a combination with vincristine on EwS cells, while this drug pair exhibits antagonistic effects towards normal cells. Finally, animal studies validated that ML111-NP efficiently accumulate in orthotopic EwS xenografts after intravenous injection and provide superior therapeutic outcomes in a combination with vincristine without evident toxicity. These results support the potential of the ML111-based combinatorial therapy for EwS.

A de novo substitution in BCL11B leads to loss of interaction with transcriptional complexes and craniosynostosis.

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.

Transcription Factor CTIP1/ BCL11A Regulates Epidermal Differentiation and Lipid Metabolism During Skin Development.

The epidermal permeability barrier (EPB) prevents organisms from dehydration and infection. The transcriptional regulation of EPB development is poorly understood. We demonstrate here that transcription factor COUP-TF-interacting protein 1 (CTIP1/BCL11A; hereafter CTIP1) is highly expressed in the developing murine epidermis. Germline deletion of Ctip1 (Ctip1 -/-) results in EPB defects accompanied by compromised epidermal differentiation, drastic reduction in profilaggrin processing, reduced lamellar bodies in granular layers and significantly altered lipid composition. Transcriptional profiling of Ctip1 -/- embryonic skin identified altered expression of genes encoding lipid-metabolism enzymes, skin barrier-associated transcription factors and junctional proteins. CTIP1 was observed to interact with genomic elements within the regulatory region of the gene encoding the differentiation-associated gene, Fos-related antigen2 (Fosl2) and lipid-metabolism-related gene, Fatty acid elongase 4 (Elvol4), and the expression of both was altered in Ctip1 -/- mice. CTIP1 appears to play a role in EPB establishment of via direct or indirect regulation of a subset of genes encoding proteins involved in epidermal differentiation and lipid metabolism. These results identify potential, CTIP1-regulated avenues for treatment of skin disorders involving EBP defects.

Cytotoxic effects of peanut phenolics possessing histone deacetylase inhibitory activity in breast and cervical cancer cell lines.

BACKGROUND: Epigenetic histone modifications are considered as a promising avenue for cancer preventive and therapeutic strategies. The purpose of this study was to evaluate the antiproliferative and histone deacetylase (HDAC) inhibitory activity of selected peanut phenolics, including p-coumaric acid, ferulic acid, sinapinic acid and resveratrol, in MCF-7 and HeLa cells. METHODS: The cytotoxic and HDAC inhibitory activities were assessed by MTT assays, flow cytometric analyses of cell cycle arrest and apoptosis induction, and western blotting. RESULTS: The results showed that all four phenolics inhibited proliferation of both MCF-7 and HeLa cells in a dose-dependent manner. Among the phenolics tested, resveratrol was the most effective in inhibiting growth of cancer cells. Treatment with all phenolics resulted in histone H3 hyperacetylation in both cell lines, indicating potential for HDAC inhibition. These phenolics induced apoptosis in both MCF-7 and HeLa cells in a concentration-dependent manner. Moreover, all phenolics induced G0/G1-phase arrest of the cell cycle in MCF-7 cells while p-coumaric and ferulic acids caused S-phase arrest in HeLa cells. Exposure to p-coumaric acid increased p53 and p21 expression but decreased CDK4 levels in both cell types, which could result in the observed G0/G1 arrest. Moreover, inhibition of ERK1/2 phosphorylation by ferulic acid and resveratrol contributed to cell growth inhibition. CONCLUSION: Peanut phenolics appear to influence the extent of histone acetylation in MCF-7 and HeLa cells, and this activity modulates multiple pathways that are implicated in cancer prevention.

BCL11B regulates sutural patency in the mouse craniofacial skeleton.

The transcription factor BCL11B plays essential roles during development of the immune, nervous, and cutaneous systems. Here we show that BCL11B is expressed in both osteogenic and sutural mesenchyme of the developing craniofacial complex. Bcl11b(-/-) mice exhibit increased proliferation of osteoprogenitors, premature osteoblast differentiation, and enhanced skull mineralization leading to synostoses of facial and calvarial sutures. Ectopic expression of Fgfr2c, a gene implicated in craniosynostosis in mice and humans, and that of Runx2 was detected within the affected sutures of Bcl11b(-/-) mice. These data suggest that ectopic expression of Fgfr2c in the sutural mesenchyme, without concomitant changes in the expression of FGF ligands, appears to induce the RUNX2-dependent osteogenic program and craniosynostosis in Bcl11b(-/-) mice.

Transcription Factor CTIP2 Maintains Hair Follicle Stem Cell Pool and Contributes to Altered Expression of LHX2 and NFATC1.

Transcription factor CTIP2 (chicken ovalbumin upstream promoter transcription factor-interacting protein 2), also known as BCL11B, is expressed in hair follicles (HFs) of embryonic and adult skin. Ctip2-null mice exhibit reduced HF density during embryonic development. In contrast, conditional inactivation of Ctip2 in the epidermis (Ctip2(ep-/-) mice) leads to a shorter telogen and a premature entry into anagen during the second phase of hair cycling without a detectable change in the number of HFs. Keratinocytes of the bulge stem cells (SCs) niche of Ctip2(ep-/-) mice proliferate more and undergo reduced apoptosis compared with the corresponding cells of wild-type mice. However, premature activation of follicular SCs in mice lacking CTIP2 leads to the exhaustion of this SC compartment in comparison with Ctip2(L2/L2) mice, which retained quiescent follicle SCs. CTIP2 modulates expression of genes encoding EGFR and NOTCH1 during formation of HFs and those encoding nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1 and LIM homeobox 2 during normal hair cycling in adult skin. The expression of most of these genes is disrupted in mice lacking CTIP2, and these alterations may underlie the phenotype of Ctip2-null and Ctip2(ep-/-) mice. CTIP2 appears to serve as a transcriptional organizer that integrates input from multiple signaling cues during HF morphogenesis and hair cycling.

Kinetic analysis of BCL11B multisite phosphorylation-dephosphorylation and coupled sumoylation in primary thymocytes by multiple reaction monitoring mass spectroscopy.

Transcription factors with multiple post-translational modifications (PTMs) are not uncommon, but comprehensive information on site-specific dynamics and interdependence is comparatively rare. Assessing dynamic changes in the extent of PTMs has the potential to link multiple sites both to each other and to biological effects observable on the same time scale. The transcription factor and tumor suppressor BCL11B is critical to three checkpoints in T-cell development and is a target of a T-cell receptor-mediated MAP kinase signaling. Multiple reaction monitoring (MRM) mass spectroscopy was used to assess changes in relative phosphorylation on 18 of 23 serine and threonine residues and sumoylation on one of two lysine resides in BCL11B. We have resolved the composite phosphorylation-dephosphorylation and sumoylation changes of BCL11B in response to MAP kinase activation into a complex pattern of site-specific PTM changes in primary mouse thymocytes. The site-specific resolution afforded by MRM analyses revealed four kinetic patterns of phosphorylation and one of sumoylation, including both rapid simultaneous site-specific increases and decreases at putative MAP kinase proline-directed phosphorylation sites, following stimulation. These data additionally revealed a novel spatiotemporal bisphosphorylation motif consisting of two kinetically divergent proline-directed phosphorylation sites spaced five residues apart.

Grp1-associated scaffold protein regulates skin homeostasis after ultraviolet irradiation.

Grp1-associated scaffold protein (Grasp), the product of a retinoic acid-induced gene in P19 embryonal carcinoma cells, is expressed primarily in brain, heart, and lung of the mouse. We report herein that Grasp transcripts are also found in mouse skin in which the Grasp gene is robustly induced following acute ultraviolet-B (UVB) exposure. Grasp(-/-) mice were found to exhibit delayed epidermal proliferation and a blunted apoptotic response after acute UVB exposure. Immunohistochemical analyses revealed that the nuclear residence time of the tumor suppressor protein p53 was reduced in Grasp(-/-) mice after UVB exposure. Taken together, our results suggest that a physiological role of Grasp may be to regulate skin homeostasis after UVB exposure, potentially by influencing p53-mediated apoptotic responses in skin.

Regulation of transcription factor activity by interconnected post-translational modifications.

Transcription factors comprise just over 7% of the human proteome and serve as gatekeepers of cellular function, integrating external signal information into gene expression programs that reconfigure cellular physiology at the most basic levels. Surface-initiated cell signaling pathways converge on transcription factors, decorating these proteins with an array of post-translational modifications (PTMs) that are often interdependent, being linked in time, space, and combinatorial function. These PTMs orchestrate every activity of a transcription factor over its entire lifespan--from subcellular localization to protein-protein interactions, sequence-specific DNA binding, transcriptional regulatory activity, and protein stability--and play key roles in the epigenetic regulation of gene expression. The multitude of PTMs of transcription factors also offers numerous potential points of intervention for development of therapeutic agents to treat a wide spectrum of diseases. We review PTMs most commonly targeting transcription factors, focusing on recent reports of sequential and linked PTMs of individual factors.

Transcription factor Ctip2 controls epidermal lipid metabolism and regulates expression of genes involved in sphingolipid biosynthesis during skin development.

The stratum corneum is composed of protein-enriched corneocytes embedded in an intercellular matrix of nonpolar lipids organized as lamellar layers and giving rise to epidermal permeability barrier (EPB). EPB defects have an important role in the pathophysiology of skin diseases such as eczema. The transcriptional control of skin lipid metabolism is poorly understood. We have discovered that mice lacking transcription factor COUP-TF-interacting protein 2 (Ctip2) exhibit EPB defects including altered keratinocyte terminal differentiation, delayed skin barrier development, and interrupted neutral lipid distribution in the epidermis. Here we adapted a targeted lipidomic approach using mass spectrometry and have determined that Ctip2(-/-) mice (germline deletion of the Ctip2 gene) display altered composition of major epidermal lipids, such as ceramides and sphingomyelins, compared with wild-type mice at different stages of skin development. Interestingly, expressions of several genes involved in skin sphingolipid biosynthesis and metabolism were altered in mutant skin. Ctip2 was found to be recruited to the promoter region of a subset of those genes, suggesting their possible direct regulation by Ctip2. Our results confirm an important role of Ctip2 in regulating skin lipid metabolism and indicate that profiling of epidermal sphingolipid could be useful for designing effective strategies to improve barrier dysfunctions.

Ctip2 is a dynamic regulator of epidermal proliferation and differentiation by integrating EGFR and Notch signaling.

Epidermal morphogenesis results from a delicate balance between keratinocyte proliferation and differentiation, and this balance is perturbed upon deletion of transcription factor Ctip2. Here we demonstrate that Ctip2, in a cell autonomous manner, controls keratinocyte proliferation and cytoskeletal organization, and regulates the onset and maintenance of differentiation in keratinocytes in culture. Ctip2 integrates keratinocyte proliferation and the switch to differentiation by directly and positively regulating EGFR transcription in proliferating cells and Notch1 transcription in differentiating cells. In proliferative cells, the EGFR promoter is occupied by Ctip2, whereas Ctip2 is only recruited to the Notch1 promoter under differentiating conditions. Activation of EGFR signaling downregulates Ctip2 at the transcript level, whereas high calcium signaling triggers SUMOylation, ubiquitination and proteasomal degradation of Ctip2 at the protein level. Together, our findings demonstrate a novel mechanism(s) of Ctip2-mediated, coordinated control of epidermal proliferation and terminal differentiation, and identify a pathway of negative feedback regulation of Ctip2 during epidermal development.

Grp1-associated scaffold protein (GRASP) is a regulator of the ADP ribosylation factor 6 (Arf6)-dependent membrane trafficking pathway.

GRASP interacts with Grp1 (general receptor for phosphoinositides 1; cytohesin 3), which catalyses nucleotide exchange on and activation of Arf6 (ADP-ribosylation factor-6). Arf6 is a low-molecular-mass GTPase that regulates key aspects of endocytic recycling pathways. Overexpressed GRASP accumulated in the juxtanuclear ERC (endocytic recycling compartment). GRASP co-localized with a constitutively inactive mutant of Arf6 in the ERC such that it was reversed by expression of wild-type Grp1. Co-expression of GRASP and Grp1 promoted membrane ruffling, a cellular hallmark of Arf6 activation. GRASP accumulation in ERC was found to block recycling of the MHC-I (major histocompatibility complex-I), which is trafficked by the Arf6-dependent pathway. In contrast, overexpression of GRASP had no effect on the recycling of transferrin receptors, which are trafficked by a clathrin-dependent pathway. The findings suggest that GRASP regulates the non-clathrin/Arf6-dependent, plasma membrane recycling and signalling pathways.

Coordinated regulation of transcription factor Bcl11b activity in thymocytes by the mitogen-activated protein kinase (MAPK) pathways and protein sumoylation.

The transcriptional regulatory protein Bcl11b is essential for T-cell development. We have discovered a dynamic, MAPK-regulated pathway involving sequential, linked, and reversible post-translational modifications of Bcl11b in thymocytes. MAPK-mediated phosphorylation of Bcl11b was coupled to its rapid desumoylation, which was followed by a subsequent cycle of dephosphorylation and resumoylation. Additionally and notably, we report the first instance of direct identification by mass spectrometry of a site of small ubiquitin-like modifier (SUMO) adduction, Lys-679 of Bcl11b, in a protein isolated from a native, mammalian cell. Sumoylation of Bcl11b resulted in recruitment of the transcriptional co-activator p300 to a Bcl11b-repressed promoter with subsequent induction of transcription. Prolonged treatment of native thymocytes with phorbol 12,13-dibutyrate together with the calcium ionophore A23187 also promoted ubiquitination and proteasomal degradation of Bcl11b, providing a mechanism for signal termination. A Bcl11b phospho-deSUMO switch was identified, the basis of which was phosphorylation-dependent recruitment of the SUMO hydrolase SENP1 to phospho-Bcl11b, coupled to hydrolysis of SUMO-Bcl11b. These results define a regulatory pathway in thymocytes that includes the MAPK pathways and upstream signaling components, Bcl11b and the associated nucleosome remodeling and deacetylation (NuRD) complex, SENP proteins, the Bcl11b protein phosphatase 6, the sumoylation machinery, the histone acetyltransferase p300, and downstream transcriptional machinery. This pathway appears to facilitate derepression of repressed Bcl11b target genes as immature thymocytes initiate differentiation programs, biochemically linking MAPK signaling with the latter stages of T-cell development.

BCL11B regulates epithelial proliferation and asymmetric development of the mouse mandibular incisor.

Mouse incisors grow continuously throughout life with enamel deposition uniquely on the outer, or labial, side of the tooth. Asymmetric enamel deposition is due to the presence of enamel-secreting ameloblasts exclusively within the labial epithelium of the incisor. We have previously shown that mice lacking the transcription factor BCL11B/CTIP2 (BCL11B hereafter) exhibit severely disrupted ameloblast formation in the developing incisor. We now report that BCL11B is a key factor controlling epithelial proliferation and overall developmental asymmetry of the mouse incisor: BCL11B is necessary for proliferation of the labial epithelium and development of the epithelial stem cell niche, which gives rise to ameloblasts; conversely, BCL11B suppresses epithelial proliferation, and development of stem cells and ameloblasts on the inner, or lingual, side of the incisor. This bidirectional action of BCL11B in the incisor epithelia appears responsible for the asymmetry of ameloblast localization in developing incisor. Underlying these spatio-specific functions of BCL11B in incisor development is the regulation of a large gene network comprised of genes encoding several members of the FGF and TGFß superfamilies, Sprouty proteins, and Sonic hedgehog. Our data integrate BCL11B into these pathways during incisor development and reveal the molecular mechanisms that underlie phenotypes of both Bcl11b(-/-) and Sprouty mutant mice.

Determination of gene expression patterns by whole-mount in situ hybridization.

Whole-mount in situ hybridization (WISH) is a reliable and specific method to study three-dimensional patterns of gene expression. A labeled nucleic acid probe anneals to a complementary target sequence and is visualized and localized in the embryo. This chapter describes a sensitive method for WISH on mouse embryos using digoxigenin-labeled RNA probes. The technique can be used for the analysis of gene expression patterns during early stages of odontogenesis and in tooth explants.

Determination of gene expression patterns by in situ hybridization in sections.

In recent years, in situ RNA hybridization technique has found a widespread application in developmental biology. This method has frequently been used to determine gene expression patterns, which is a first step toward understanding of a gene function. Here, we provide a reliable and sensitive method for in situ RNA hybridization on frozen sections of mouse embryo using digoxigenin-labeled RNA probes. This technique can be used to study gene expression patterns at all stages of odontogenesis.