The Stat3/GR interaction code: predictive value of direct/indirect DNA recruitment for transcription outcome.

Transcription factor recruitment to genomic sites of action is primarily due to direct protein:DNA interactions. The subsequent recruitment of coregulatory complexes leads to either transcriptional activation or repression. In contrast to this canonical scheme, some transcription factors, such as the glucocorticoid receptor (GR), behave as transcriptional repressors when recruited to target genes through protein tethering. We have investigated the genome-wide prevalence of tethering between GR and Stat3 and found nonreciprocal interactions, namely that GR tethering to DNA-bound Stat3 results in transcriptional repression, whereas Stat3 tethering to GR results in synergism. Further, other schemes of GR and Stat3 corecruitment to regulatory modules result in transcriptional synergism, including neighboring and composite binding sites. The results indicate extensive transcriptional interactions between Stat3 and GR; further, they provide a genome-wide assessment of transcriptional regulation by tethering and a molecular basis for integration of signals mediated by GR and Stats in health and disease.

A comparative study of ChIP-seq sequencing library preparation methods.

BACKGROUND: ChIP-seq is the primary technique used to investigate genome-wide protein-DNA interactions. As part of this procedure, immunoprecipitated DNA must undergo "library preparation" to enable subsequent high-throughput sequencing. To facilitate the analysis of biopsy samples and rare cell populations, there has been a recent proliferation of methods allowing sequencing library preparation from low-input DNA amounts. However, little information exists on the relative merits, performance, comparability and biases inherent to these procedures. Notably, recently developed single-cell ChIP procedures employing microfluidics must also employ library preparation reagents to allow downstream sequencing. RESULTS: In this study, seven methods designed for low-input DNA/ChIP-seq sample preparation (Accel-NGS® 2S, Bowman-method, HTML-PCR, SeqPlex™, DNA SMART™, TELP and ThruPLEX®) were performed on five replicates of 1 ng and 0.1 ng input H3K4me3 ChIP material, and compared to a "gold standard" reference PCR-free dataset. The performance of each method was examined for the prevalence of unmappable reads, amplification-derived duplicate reads, reproducibility, and for the sensitivity and specificity of peak calling. CONCLUSIONS: We identified consistent high performance in a subset of the tested reagents, which should aid researchers in choosing the most appropriate reagents for their studies. Furthermore, we expect this work to drive future advances by identifying and encouraging use of the most promising methods and reagents. The results may also aid judgements on how comparable are existing datasets that have been prepared with different sample library preparation reagents.

The Ets factor Etv1 interacts with Tpit protein for pituitary pro-opiomelanocortin (POMC) gene transcription.

Pro-opiomelanocortin (POMC) is expressed in two lineages of the pituitary, the anterior lobe corticotrophs and the intermediate lobe melanotrophs. POMC expression in these two lineages is highly dependent on the cell-restricted transcription factor Tpit. As Tpit intervenes relatively late in differentiation of those lineages, we have been searching for other transcription factors that may participate in their gene expression program. On the basis of similarity with the Tpit expression profile, we identified Ets variant gene 1 (Etv1/Er81) as a putative POMC transcription factor. Using Etv1-lacZ knockin mice, we describe preferential Etv1 expression in pituitary POMC cells and also in posterior lobe pituicytes. We further show that Etv1 enhances POMC transcription on its own and in synergy with Tpit. The Ets-binding site located within the Tpit/Pitx regulatory element is necessary for Etv1 activity in POMC-expressing AtT-20 cells but dispensable for synergy with Tpit. Etv1 and Tpit interact together in coimmunoprecipitation experiments. Furthermore, Etv1 is present at the POMC promoter, and siRNA-mediated knockdown of Etv1 in AtT-20 cells produces a significant decrease in POMC expression. Etv1 knockout pituitaries show normal POMC cell distribution and normal POMC mRNA abundance, suggesting compensation by other factors. The coordinate expression of Etv1 with POMC cell differentiation and its interaction with the highly cell-restricted Tpit factor indicate that Etv1 participates in a combinatorial code for pituitary cell-specific gene expression.

Regulatory network analyses reveal genome-wide potentiation of LIF signaling by glucocorticoids and define an innate cell defense response.

While the hypothalamo-pituitary-adrenal axis (HPA) activates a general stress response by increasing glucocorticoid (Gc) synthesis, biological stress resulting from infections triggers the inflammatory response through production of cytokines. The pituitary gland integrates some of these signals by responding to the pro-inflammatory cytokines IL6 and LIF and to a negative Gc feedback loop. The present work used whole-genome approaches to define the LIF/STAT3 regulatory network and to delineate cross-talk between this pathway and Gc action. Genome-wide ChIP-chip identified 3,449 STAT3 binding sites, whereas 2,396 genes regulated by LIF and/or Gc were found by expression profiling. Surprisingly, LIF on its own changed expression of only 85 genes but the joint action of LIF and Gc potentiated the expression of more than a thousand genes. Accordingly, activation of both LIF and Gc pathways also potentiated STAT3 and GR recruitment to many STAT3 targets. Our analyses revealed an unexpected gene cluster that requires both stimuli for delayed activation; 83% of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response.

Content of client emails in internet-delivered cognitive behaviour therapy: A comparison between two trials and relationship to client outcome.

Many Internet-delivered cognitive behavioural therapy (ICBT) programs include email communication between clients and therapists as a part of treatment; yet relatively little is known about the nature and impact of this communication. Previous research conducted by Svartvatten et al. (2015) has identified 10 themes in written correspondence by clients accessing ICBT for depression. The current study examined: (1) if previously identified themes in client emails would be present in a shorter ICBT program for depression and anxiety; and (2) whether themes in emails similarly correlated with symptom improvement, lesson completion, and perceptions of working alliance. Using 80 randomly selected clients from a published ICBT trial (ISRCTN42729166; Hadjistavropoulos et al., 2016), client emails (average 5.69 per client) were examined for the presence of the themes reported by Svartvatten et al. (2015) and correlated with symptom improvement, lesson completion, perceptions of working alliance. Although most themes developed by Svartvatten et al. (2015) were identified in client emails, the frequency of themes differed between studies. Most notably, emails in the current study were more often coded as involving alliance bolstering (~39% vs. 22% of statements) and identification of patterns and problem behaviours (~25% vs. 6% of statements). Greater frequency of tries alternative behaviour and identifies patterns and problem behaviours were correlated with a greater number of lessons completed. In terms of symptom change, greater frequency of maladaptive repetitive thinking and problems with treatment content in the emails were correlated with smaller improvements in anxiety, whereas observes positive consequences was correlated with larger improvements in anxiety. Similarly, greater frequency of maladaptive repetitive thinking was correlated with smaller improvements in depression. Regarding perceptions of working alliance, more frequent statements of observes positive consequences was correlated with higher alliance. The research provides clinicians and researchers with an improved understanding of the comparability and meaning of client communication in different ICBT programs. Experimental research is needed to better understand the role of client communication in ICBT.

The TPIT gene mutation M86R associated with isolated adrenocorticotropin deficiency interferes with protein: protein interactions.

CONTEXT: Tpit is a T-box transcription factor important for terminal differentiation of pituitary proopiomelanocortin-expressing cells. We previously showed that human and murine mutations in the gene encoding this highly cortico/melanotrope-specific transcription factor cause a neonatal onset form of congenital isolated ACTH deficiency (IAD). We characterized the largest series of neonatal IAD patients caused by TPIT mutations, and this revealed a highly homogeneous clinical presentation. So far, 12 different loss-of-function TPIT mutations have been identified. The methionine 86 arginine (M86R) TPIT mutation was recently identified in compound heterozygosity with the 782delA frame-shift mutation in two siblings with early-onset IAD. OBJECTIVE: We conducted a functional analysis of the missense M86R mutation to assess transcriptional activity, DNA binding activity, and nuclear location, as well as protein-protein interactions. RESULTS: Although the M86 residue is located within the T-box DNA-binding domain, it did not affect monomer DNA-binding activity per se, but it impaired DNA binding with other DNA-bound proteins, including itself (homodimers) and pituitary homeobox 1 (Pitx1). The M86 residue is at the interface between T domains in the T dimers crystal structure, and it appears that the same residue is involved in heterodimer formation with pituitary Pitx1. Furthermore, TPIT M86R is deficient in the recruitment of the coactivator SRC2 that partly mediates the CRH stimulation of proopiomelanocortin transcription. CONCLUSION: Thus, the M86R TPIT mutation is defining an important surface of the T domain for multiple protein interactions and for transcription.

The Cables1 Gene in Glucocorticoid Regulation of Pituitary Corticotrope Growth and Cushing Disease.

CONTEXT: Cushing disease (CD) is due to pituitary corticotrope adenomas that produce unrestrained ACTH secretion and have lost the negative feedback exerted by glucocorticoids (GCs). GCs also restrain corticotrope proliferation, and the mechanisms of this inhibition are poorly understood. OBJECTIVE: The aim of the study was to identify cell cycle regulatory genes that are regulated by GCs and the glucocorticoid receptor and to assess regulatory genes that have a rate-limiting action on corticotrope proliferation and may be disregulated in CD. DESIGN: The mouse corticotrope tumor cells AtT-20 were used to identify GC-regulated genes that contribute to control of cell cycle progression. Surgery sections from patients with CD were used to assess expression of CABLES1 in corticotrope adenomas. METHODS: Gene expression profiling, small interfering RNA knockdowns, cell cycle analyses, and genetic manipulations were performed in AtT-20 cells. Sequencing of chromatin immunoprecipitation for pituitary-restricted transcription factors and RNA polymerase II were used to identify regulatory elements and genes that bind GR and are direct transcriptional targets. A panel of previously well-characterized corticotrope adenomas was used to correlate expression of CABLES1 with that of other markers. RESULTS: GCs altered expression of 3 positive and 3 negative regulators of cell cycle progression. Two Myc genes (L-Myc and N-Myc) and E2F2 are repressed by GCs, whereas genes for the negative regulators of the cell cycle, Gadd45ß, Gadd45γ, and Cables1 are activated by GCs. Cables1 small interfering RNA knockdown strongly stimulates AtT-20 cell proliferation and antagonizes the growth inhibition produced by GCs. The Gadd45 and Cables1 genes have the hallmarks of direct GC targets. CABLES1 is expressed in normal human pituitary cells, but expression is lost in ∼55% of corticotrope adenomas, and this is strongly correlated with the loss of p27(Kip1) expression. CONCLUSIONS: CABLES1 is a critical regulator of corticotrope proliferation that defines a pathway often inactivated in CD and links proliferation to GC resistance.

[Tpit mutations reveal a new model of pituitary differentiation and account for isolated ACTH deficiency]. / Mutations du facteur de transcription Tpit et différenciation hypophysaire.

Pituitary hormone-producing cells differentiate sequentially from a common epithelial primordium, Rathke's pouch, under the combinatorial action of a subset of tissue- and cell-restricted transcription factors. Some factors have been implicated in early events of pituitary induction and morphogenesis while other factors like Pit-1 and SF-1 have been associated with differentiation of particular lineages. In POMC-expressing cells, Pitx1, NeuroD1 and Tpit were shown to be important for cell specific transcription of the POMC gene. Since Tpit is exclusively expressed in pituitary POMC-expressing lineages, the corticotrophs and melanotrophs, we investigated the TPIT gene coding sequences in 17 patients presenting with congenital isolated ACTH deficiency (IAD). We demonstrated that human TPIT gene mutations cause a neonatal onset form of IAD (8/11), but not juvenile forms of this deficiency (0/6). In the absence of glucocorticoid replacement, IAD can lead to neonatal death by acute adrenal insufficiency. To assess the importance of Tpit in pituitary differentiation and function, we produced Tpit-null mice. Concordant with the human phenotype, Tpit-null mice have IAD : plasma ACTH is greatly reduced in these mice, their plasma corticosterone is undetectable and the adrenals are hypoplastic. Analysis of the pituitary in Tpit-null mice revealed multiple roles of this factor in cell differentiation. First, Tpit is a positive regulator for POMC cell differentiation. Tpit is also a negative regulator of the pituitary gonadotroph fate. Thus, Tpit operates as a molecular switch to orient differentiation of a common precursor towards either POMC or gonadotroph fate. A binary choice model of pituitary cell differentiation is presented.

Adult pituitary cell maintenance: lineage-specific contribution of self-duplication.

The identification of a stable pool of progenitor/stem cells in the adult pituitary has renewed the interest of identifying mechanisms for maintenance of pituitary cells throughout life. Whereas developmental studies have shown that progenitor expansion is the major source of new differentiated cells during pituitary organogenesis, the contribution of these progenitors for maintenance of the adult tissue is not clear although progenitors were clearly involved in cell expansion following end-organ ablation, notably after adrenalectomy and/or gonadectomy. We have used a genetic trick that eliminates dividing cells by apoptosis in order to assess the contribution of differentiated corticotropes and melanotropes for maintenance of their population in the adult pituitary. The system relies on chromosome instability created by the action of the Cre recombinase on inverted loxP sites. Expression of Cre recombinase in corticotropes and melanotropes led to progressive loss of corticotropes whereas melanotropes were unaffected. Because the Cre transgene is not expressed in progenitors, the data indicate that maintenance of the adult corticotrope pool is primarily due to self-duplication of differentiated cells. In contrast, melanotropes do not divide. Maintenance of corticotropes by self-duplication contrasts with the reported proliferative response of undifferentiated cells observed after adrenalectomy. If corticotrope reentry into cell cycle constitutes a normal mechanism to maintain the adult corticotrope pool, this same mechanism may also be perturbed during corticotrope adenoma development in Cushing's disease.

A pituitary-specific enhancer of the POMC gene with preferential activity in corticotrope cells.

Cell-specific expression of the pituitary proopiomelanocortin (POMC) gene depends on the combination of tissue- and cell-restricted transcription factors such as Pitx1 and Tpit. These factors act on the proximal POMC promoter together with transcription factors that integrate inputs from signaling pathways. We now report the identification of an upstream enhancer in the POMC locus that is targeted by the same subset of transcription factors, except Pitx1. This enhancer located at -7 kb in the mouse POMC gene is highly dependent on Tpit for activity. Whereas Tpit requires Pitx1 for action on the promoter, it acts on the -7-kb enhancer as homodimers binding to a palindromic Tpit response element (TpitRE). Both half-sites of the TpitRE palindrome and Tpit homodimerization are required for activity. In vivo, the enhancer exhibits preferential activity in corticotrope cells of the anterior lobe whereas the promoter exhibits preference for intermediate lobe melanotropes. The enhancer is conserved among different species with the TpitRE palindrome localized at the center of conserved sequences. However, the mouse and human -7-kb enhancers do not exhibit conservation of hormone responsiveness and may differ in their relative importance for POMC expression. In summary, pituitary expression of the POMC gene relies on an upstream enhancer that complements the activity of the proximal promoter with Tpit as the major regulator of both regulatory regions.

The T-box factor Tpit recruits SRC/p160 co-activators and mediates hormone action.

Tpit (Tbx19) is a transcription factor belonging to the T-box family, and it is essential for late differentiation of pituitary pro-opiomelanocortin (POMC)-expressing corticotroph and melanotroph cells. Tpit is also required, both in humans and mice, for cell-specific expression of the POMC gene in cooperation with the homeoprotein Pitx1. Despite their important roles as developmental regulators, the molecular mechanisms underpinning the functions of T-box factors in general, and of Tpit in particular, are still poorly defined. We now report that Tpit functions as an activator of transcription by recruiting SRC/p160 co-activators to its cognate DNA target in the POMC promoter, the Tpit/Pitx-RE. We also show that Tpit is a mediator of hormone signaling and that the Tpit/Pitx-RE is responsive to signals elicited by hypothalamic corticotropin-releasing hormone. These signals are mediated by the cAMP-dependent protein kinase and mitogen-activated protein kinase pathways, and activation of cAMP-dependent protein kinase also enhances Tpit and SRC-dependent transcription. We have previously shown that corticotropin-releasing hormone action is also exerted at the POMC promoter through the orphan nuclear receptor NGFI-B and its recruitment of SRC co-activators. Given that Tpit exhibits transcriptional synergy with NGFI-B, our results suggest that Tpit, along with NGFI-B and SRC-2, is part of a transcription regulatory complex assembled on the POMC promoter in response to hormonal stimulation.

Tpit determines alternate fates during pituitary cell differentiation.

The T-box transcription factor Tpit was identified as a cell-specific factor for expression of the pituitary proopiomelanocortin (POMC) gene. Expression of this factor is exclusively restricted to the pituitary POMC-expressing lineages, the corticotrophs and melanotrophs. We have now determined the role of this factor in pituitary cell differentiation. Tpit is a positive regulator for late POMC cell differentiation and POMC expression, but it is not essential for lineage commitment. The pituitary intermediate lobe normally contains only Tpit-expressing melanotrophs. Inactivation of the Tpit gene results in almost complete loss of POMC-expressing cells in this tissue, which now has a large number of gonadotrophs and a few clusters of Pit-1-independent thyrotrophs. The role of Tpit as a negative regulator of gonadotroph differentiation was confirmed in transgenic gain-of-function experiments. One mechanism to account for the negative role of Tpit in differentiation may be trans-repression between Tpit and the gonadotroph-restricted factor SF1. These data suggest that antagonism between Tpit and SF1 may play a role in establishment of POMC and gonadotroph lineages and that these lineages may arise from common precursors.

Human and mouse TPIT gene mutations cause early onset pituitary ACTH deficiency.

Tpit is a highly cell-restricted transcription factor that is required for expression of the pro-opiomelanocortin (POMC) gene and for terminal differentiation of the pituitary corticotroph lineage. Its exclusive expression in pituitary POMC-expressing cells has suggested that its mutation may cause isolated deficiency of pituitary adrenocorticotropin (ACTH). We now show that Tpit-deficient mice constitute a model of isolated ACTH deficiency (IAD) that is very similar to human IAD patients carrying TPIT gene mutations. Through genetic analysis of a panel of IAD patients, we show that TPIT gene mutations are associated at high frequency with early onset IAD, but not with juvenile forms of this deficiency. We identified seven different TPIT mutations, including nonsense, missense, point deletion, and a genomic deletion. This work defines congenital early onset IAD as a relatively homogeneous clinical entity caused by recessive transmission of loss-of-function mutations in the TPIT gene.