Pbx Regulates Patterning of the Cerebral Cortex in Progenitors and Postmitotic Neurons

SummaryWe demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2+/− sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex, ventral expansion of the dorsomedial cortex, and ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate

Transcriptional Regulation of Enhancers Active in Protodomains of the Developing Cerebral Cortex

SummaryElucidating the genetic control of cerebral cortical (pallial) development is essential for understanding function, evolution, and disorders of the brain. Transcription factors (TFs) that embryonically regulate pallial regionalization are expressed in gradients, raising the question of how discrete domains are generated. We provide evidence that small enhancer elements active in protodomains integrate broad transcriptional information. CreERT2 and GFP expression from 14 different enhancer elements in stable transgenic mice allowed us to define a comprehensive regional fate map of the pallium. We explored transcriptional mechanisms that control the activity of the enhancers using informatics, in vivo occupancy by TFs that regulate cortical patterning (CoupTFI, Pax6, and Pbx1), and analysis of enhancer activity in Pax6 mutants. Overall, the results provide insights into how broadly expressed patterning TFs regulate the activity of small enhancer elements that drive gene expression in pallial protodomains that fate map to distinct cortical regions

HDAC-6 inhibition ameliorates the early neuropathology in a mouse model of Krabbe disease

IntroductionIn Krabbe disease (KD), mutations in β-galactosylceramidase (GALC), a lysosomal enzyme responsible for the catabolism of galactolipids, leads to the accumulation of its substrates galactocerebroside and psychosine. This neurologic condition is characterized by a severe and progressive demyelination together with neuron-autonomous defects and degeneration. Twitcher mice mimic the infantile form of KD, which is the most common form of the human disease. The Twitcher CNS and PNS present demyelination, axonal loss and neuronal defects including decreased levels of acetylated tubulin, decreased microtubule stability and impaired axonal transport.MethodsWe tested whether inhibiting the α-tubulin deacetylase HDAC6 with a specific inhibitor, ACY-738, was able to counteract the early neuropathology and neuronal defects of Twitcher mice.ResultsOur data show that delivery of ACY-738 corrects the low levels of acetylated tubulin in the Twitcher nervous system. Furthermore, it reverts the loss myelinated axons in the sciatic nerve and in the optic nerve when administered from birth to postnatal day 9, suggesting that the drug holds neuroprotective properties. The extended delivery of ACY-738 to Twitcher mice delayed axonal degeneration in the CNS and ameliorated the general presentation of the disease. ACY-738 was effective in rescuing neuronal defects of Twitcher neurons, stabilizing microtubule dynamics and increasing the axonal transport of mitochondria.DiscussionOverall, our results support that ACY-738 has a neuroprotective effect in KD and should be considered as an add-on therapy combined with strategies targeting metabolic correction

Chemical Inhibition of Histone Deacetylases 1 and 2 Induces Fetal Hemoglobin through Activation of GATA2.

Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and β-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients. Gene expression profiling of erythroid progenitors treated with ACY-957 identified global changes in gene expression that were significantly enriched in genes previously shown to be affected by HDAC1 or 2 knockdown. These genes included GATA2, which was induced greater than 3-fold. Lentiviral overexpression of GATA2 in primary erythroid progenitors increased HBG, and reduced adult β-globin mRNA (HBB). Furthermore, knockdown of GATA2 attenuated HBG induction by ACY-957. Chromatin immunoprecipitation and sequencing (ChIP-Seq) of primary erythroid progenitors demonstrated that HDAC1 and 2 occupancy was highly correlated throughout the GATA2 locus and that HDAC1/2 inhibition led to elevated histone acetylation at well-known GATA2 autoregulatory regions. The GATA2 protein itself also showed increased binding at these regions in response to ACY-957 treatment. These data show that chemical inhibition of HDAC1/2 induces HBG and suggest that this effect is mediated, at least in part, by histone acetylation-induced activation of the GATA2 gene

Bcl11b represses a mature T-cell gene expression program in immature CD4(+)CD8(+) thymocytes

Bcl11b is a transcription factor that, within the hematopoietic system, is expressed specifically in T cells. Although Bcl11b is required for T-cell differentiation in newborn Bcl11b-null mice, and for positive selection in the adult thymus of mice bearing a T-cell-targeted deletion, the gene network regulated by Bcl11b in T cells is unclear. We report herein that Bcl11b is a bifunctional transcriptional regulator, which is required for the correct expression of approximately 1000 genes in CD4(+)CD8(+)CD3(lo) double-positive (DP) thymocytes. Bcl11b-deficient DP cells displayed a gene expression program associated with mature CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes, including upregulation of key transcriptional regulators, such as Zbtb7b and Runx3. Bcl11b interacted with regulatory regions of many dysregulated genes, suggesting a direct role in the transcriptional regulation of these genes. However, inappropriate expression of lineage-associated genes did not result in enhanced differentiation, as deletion of Bcl11b in DP cells prevented development of SP thymocytes, and that of canonical NKT cells. These data establish Bcl11b as a crucial transcriptional regulator in thymocytes, in which Bcl11b functions to prevent the premature expression of genes fundamental to the SP and NKT cell differentiation programs

Dual role of COUP-TF-interacting protein 2 in epidermal homeostasis and permeability barrier formation.

International audienceCOUP-TF-interacting protein 2 (CTIP2; also known as Bcl11b) is a transcription factor that plays key roles in the development of the central nervous and immune systems. CTIP2 is also highly expressed in the developing epidermis, and at lower levels in the dermis and in adult skin. Analyses of mice harboring a germline deletion of CTIP2 revealed that the protein plays critical roles in skin during development, particularly in keratinocyte proliferation and late differentiation events, as well as in the development of the epidermal permeability barrier. At the core of all of these actions is a relatively large network of genes, described herein, that is regulated directly or indirectly by CTIP2. The analysis of conditionally null mice, in which expression of CTIP2 was ablated specifically in epidermal keratinocytes, suggests that CTIP2 functions in both cell and non-cell autonomous contexts to exert regulatory influence over multiple phases of skin development, including barrier establishment. Considered together, our results suggest that CTIP2 functions as a top-level regulator of skin morphogenesis

Transcriptional regulation of enhancers active in protodomains of the developing cerebral cortex.

Elucidating the genetic control of cerebral cortical (pallial) development is essential for understanding function, evolution, and disorders of the brain. Transcription factors (TFs) that embryonically regulate pallial regionalization are expressed in gradients, raising the question of how discrete domains are generated. We provide evidence that small enhancer elements active in protodomains integrate broad transcriptional information. CreER(T2) and GFP expression from 14 different enhancer elements in stable transgenic mice allowed us to define a comprehensive regional fate map of the pallium. We explored transcriptional mechanisms that control the activity of the enhancers using informatics, in vivo occupancy by TFs that regulate cortical patterning (CoupTFI, Pax6, and Pbx1), and analysis of enhancer activity in Pax6 mutants. Overall, the results provide insights into how broadly expressed patterning TFs regulate the activity of small enhancer elements that drive gene expression in pallial protodomains that fate map to distinct cortical regions

HDAC1/2 inhibition by ACY-957 leads to elevated levels of GATA2 binding at the <i>HBD</i> promoter.

<p>Changes in GATA2 binding due to ACY-957 treatment were surveyed across the 70 kb β-like globin gene cluster. Cell culture, IP with anti-GATA2, and sequencing were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g008" target="_blank">Fig 8</a>. (A) The location of each β-like globin gene and LCR hypersensitivity sites (HS1-4) are shown in the ‘Gene’ track (blue). GATA2 occupancy is shown for vehicle or ACY-957 treated primary erythroblasts (‘vehicle’ and ‘ACY-957’ tracks, black). Only 6 regions had statistically significant GATA2 binding peaks in either vehicle or ACY-957 treated cells (indicated by triangles below the ‘Gene’ track). Within the 70 kb β-like globin locus, ACY-957 treatment lead to elevated levels of GATA2 binding only at a single region, found at the <i>HBD</i> promoter. ENCODE Consortium data for K562 cells shows this <i>HBD</i> region makes long-range looping interactions with the LCR (‘5C peaks’ track, gray), is a predicted enhancer (‘Chr state’ track, gray), is a region of open chromatin (‘DNase I’ track, gray), and is a region of GATA2 and GATA1 binding (‘GATA2’ and ‘GATA1’ tracks, gray). (B) Confirmation of GATA2 ChIP-Seq results by ChIP-QPCR. Two unique primer sets were used to detect GATA2 occupancy at the <i>HBD</i> promoter. A primer set at the <i>HBB</i> promoter, predicted by ChIP-Seq as a region where GATA2 had statistically significant binding that was unaltered by ACY-957 treatment, was used as a control (mean ± SD, n = 3 QPCR replicates for each of n = 3 IP replicates). P-values were calculated using a two-tailed <i>t</i> test. ***P<0.0005 for ACY-957 compared to vehicle control. (C) Summary of findings. ACY-957 inhibits HDAC1/2 leading to elevated histone acetylation at <i>GATA2</i> enhancer regions. Increased histone acetylation promotes occupancy of GATA2 at these regulatory regions, resulting in sustained activation of GATA2 during erythroid maturation through a positive autoregulatory loop. Elevated GATA2 contributes to <i>HBG</i> induction through an unknown mechanism, but may involve increased GATA2 binding at the <i>HBD</i> promoter (dashed line).</p

Effect of HDAC1/2 inhibition on gene expression in erythroid progenitors.

<p>(A) Erythroid maturation stage of cells used in gene expression profiling experiments. BM cells were cultured in CS1 expansion media and then shifted to CS1 differentiation media for 5 days with vehicle or 1 μM ACY-957. At the end of the culture period, erythroid maturation stage was determined by flow cytometry and total RNA was isolated for analysis using Affymetrix GeneChips. Experiments were performed using cells from three independent donors. (B) Differentially expressed genes resulting from ACY-957 treatment using a filter of absolute fold change greater than 1.5 and a P-value less than 0.025. (C) Gene set enrichment analysis demonstrates that genes up-regulated by HDAC2 knockdown (‘Up in <i>HDAC2</i> KD’ gene set) are significantly overrepresented at the top of a ranked list of gene expression changes resulting from ACY-957 treatment. Significant enrichment is illustrated by the positive running enrichment score (ES) marked by the green line, normalized enrichment score (NES) = 2.5, and false discovery rate (FDR) P-value < 0.001. (D) Enrichment scores of the ‘Up in <i>HDAC1</i> KD’, ‘Up in <i>HDAC2</i> KD’, and ‘Down in <i>HDAC2</i> KD’ gene sets relative to all gene sets (2777 total) in the Molecular Signatures Database collection of Chemical and Genetic Perturbations. (E) GeneChip derived gene expression ratios of candidate <i>HBG</i> modulators following ACY-957 treatment, <i>HDAC1</i> knockdown, or <i>HDAC2</i> knockdown. Ratios expressed as treatment versus control. NS, not significant. (F) Gene expression of candidate <i>HBG</i> modulators by QPCR. BM cells were cultured in CS1 expansion media, and then shifted to CS1 differentiation media for 4 days in presence of vehicle or 1 μM ACY-957. Gene expression is shown relative to <i>ACTB</i> and normalized to day 0 (mean ± SD, n = 3 cell culture replicates). P-values were calculated for day 4 using a two-tailed <i>t</i> test. *P<0.05 and ***P<0.0005 compared to vehicle treatment. Data is representative of experiments using cells from two independent donors. (G) Induction of <i>GATA2</i> mRNA by ACY-957 in cells from the four SCD donors described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g003" target="_blank">Fig 3A</a>. P-values were calculated using a two-tailed, paired <i>t</i> test. *P<0.05 compared to vehicle treatment.</p