Ad4BP (or SF-1) has been identified as a transcription factor which regulates all the steroidogenic P450 genes in the peripheral organs, and is encoded by the mammalian homologue of Drosophila FTZ-F1 gene. mRNA coding for Ad4BP was detected in the hypothalamus and pituitary of rats by RT-PCR. Immunohistochemical analyses using an antiserum to Ad4BP in the brain and pituitary revealed that the transcription factor is expressed in nuclei of the dorsomedial part of the ventromedial hypothalamus (dmVMH) and in some subpopulation of the adenohypophysial cells. Double immunostaining of the pituitary for Ad4BP and trophic peptide hormones, FSH, TSH, and ACTH, indicated a restricted localization of Ad4BP to the gonadotroph. Disruption of the mouse Ftz-FI gene was clarified to induce severe defects in the organization of the dmVMH and the function of the pituitary gonadotroph. However, some of the dm VMH neurons and pituitary gonadotrophs persisted, which provided a sharp contrast to complete agenesis of the peripheral steroidogenic tissues (adrenal and gonads) in the mutant mouse. Additional abnormalities were seen in the ventrolateral part of VMH and dorsomedial hypothalamic nucleus, both of which do not express Ad4BP but have strong reciprocal fiber-connections with the dmVMH. Aromatase P450-containing cells in the medial preoptico-amygdaloid region, which were devoid of Ad4BP, persisted even in the brain of the gene disrupted mice. The present results clearly showed that the hypothalamic and pituitary Ad4BPs are essential to normal development of the functional VMH and gonadotroph through some mechanism distinct from that in the peripheral steroidogenic tissues. 8 1995 Wiley-Liss, Inc.

In mammals, the Y-linked sex-determining gene Sry cell-autonomously promotes Sertoli cell differentiation from bipotential supporting cell precursors through SRY-box containing gene 9 (Sox9), leading to testis formation. Without Sry action, the supporting cells differentiate into granulosa cells, resulting in ovarian development. However, how Sry acts spatiotemporally to switch supporting cells from the female to the male pathway is poorly understood. We created a novel transgenic mouse line bearing an inducible Sry transgene under the control of the Hsp70.3 promoter. Analysis of these mice demonstrated that the ability of Sry to induce testis development is limited to approximately 11.0-11.25 dpc, corresponding to a time window of only 6 hours after the normal onset of Sry expression in XY gonads. If Sry was activated after 11.3 dpc, Sox9 activation was not maintained, resulting in ovarian development. This time window is delimited by the ability to engage the high-FGF9/low-WNT4 signaling states required for Sertoli cell establishment and cord organization. Our results indicate the overarching importance of Sry action in the initial 6-hour phase for the female-to-male switching of FGF9/WNT4 signaling patterns.

46,XY disorders of sex development (DSD) refer to a wide range of abnormal genitalia, including hypospadias, which affects approximately 0.5% of male newborns. We identified three different nonsense mutations of CXorf6 in individuals with hypospadias and found that its mouse homolog was specifically expressed in fetal Sertoli and Leydig cells around the critical period for sex development. These data imply that CXorf6 is a causative gene for hypospadias.

BRD4, characterized by two acetyl-lysine binding bromodomains and an extra-terminal (ET) domain, is a key chromatin organizer that directs gene activation in chromatin through transcription factor recruitment, enhancer assembly, and pause release of the RNA polymerase II complex for transcription elongation. BRD4 has been recently validated as a new epigenetic drug target for cancer and inflammation. Our current knowledge of the functional differences of the two bromodomains of BRD4, however, is limited, hindered by the lack of selective inhibitors. Here, we report our structure-guided development of diazobenzene-based small molecule inhibitors for the BRD4 bromodomains that have over 90% sequence identity at the acetyl-lysine binding site. Our lead compound MS436, through a set of water-mediated interactions, exhibits low nanomolar affinity (estimated Ki of 30–50 nM) with preference for the first bromodomain over the second. We demonstrated that MS436 effectively inhibits BRD4 activity in NF-κB-directed production of nitric oxide and pro-inflammatory cytokine interleukin-6 in murine macrophages. MS436 represents a new class of bromodomain inhibitors and will facilitate further investigation of the biological functions of the two bromodomains of BRD4 in gene expression.

As a master transcription factor in cellular responses to external stress, tumor suppressor p53 is tightly regulated. Excessive p53 activity during myocardial ischemia causes irreversible cellular injury and cardiomyocyte death. p53 activation is dependent on lysine acetylation by the lysine acetyltransferase and transcriptional co-activator CBP (CREB-binding protein) and on acetylation-directed CBP recruitment for p53 target gene expression. Here, we report a small molecule ischemin, developed with a structure-guided approach to inhibit the acetyl-lysine binding activity of the bromodomain of CBP. We show that ischemin alters post-translational modifications on p53 and histones, inhibits p53 interaction with CBP and transcriptional activity in cells, and prevents apoptosis in ischemic cardiomyocytes. Our study suggests small molecule modulation of acetylation-mediated interactions in gene transcription as a new approach to therapeutic interventions of human disorders such as myocardial ischemia.