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Chromodomain helicase domain 8 ( CHD8 ) is one of the most frequently mutated and most penetrant genes in the autism spectrum disorder (ASD). Individuals with CHD8 mutations show leading symptoms of autism, macrocephaly, and facial dysmorphisms. The molecular and cellular mechanisms underpinning the early onset and development of these symptoms are still poorly understood and prevent timely and more efficient therapies of patients. Progress in this area will require an understanding of "when, why and how cells deviate from their normal trajectories". High-throughput single-cell RNA sequencing (sc-RNAseq) directly quantifies information-bearing RNA molecules that enact each cell's biological identity. Here, we discuss recent insights from sc-RNAseq of CRISPR/Cas9-editing of Chd8/CHD8 during mouse neocorticogenesis and human cerebral organoids. Given that the deregulation of the balance between excitation and inhibition (E/I balance) in cortical and subcortical circuits is thought to represent a major etiopathogenetic mechanism in ASD, we focus on the question of whether, and to what degree, results from current sc-RNAseq studies support this hypothesis. Beyond that, we discuss the pros and cons of these approaches and further steps to be taken to harvest the full potential of these transformative techniques.

Imprinted genes play a critical role in brain development and mental health, although the underlying molecular and cellular mechanisms remain incompletely understood. The family of basic helix-loop-helix (bHLH) proteins directs the proliferation, differentiation, and specification of distinct neuronal progenitor populations. Here, we identified the bHLH factor gene Tcf4 as a direct target gene of Zac1/Plagl1, a maternally imprinted transcriptional regulator, during early neurogenesis. Zac1 and Tcf4 expression levels concomitantly increased during neuronal progenitor differentiation; moreover, Zac1 interacts with two cis-regulatory elements in the Tcf4 gene locus, and these elements together confer synergistic activation of the Tcf4 gene. Tcf4 upregulation enhances the expression of the cyclin-dependent kinase inhibitor gene p57(Kip2), a paternally imprinted Tcf4 target gene, and increases the number of cells in G1 phase. Overall, we show that Zac1 controls cell cycle arrest function in neuronal progenitors through induction of p57(Kip2) via Tcf4 and provide evidence for cooperation between imprinted genes and a bHLH factor in early neurodevelopment.

Cell-fate decisions and differentiation of embryonic and adult neural stem cells (NSC) are tightly controlled by lineage-restricted and temporal factors that interact with cell-intrinsic programs and extracellular signals through multiple regulatory loops. Imprinted genes are important players in neurodevelopment and mental health although their molecular and cellular functions remain poorly understood. Here, we show that the paternally expressed transcriptional regulator Zac1 (zinc finger protein regulating apoptosis and cell cycle arrest) is transiently induced during astroglial and neuronal differentiation of embryonic and adult NSC lines. Thereby, Zac1 transactivates Socs3 (suppressor of cytokine signaling 3), a potent inhibitor of prodifferentiative Jak/Stat3 signaling, in a lineage-specific manner to prevent precocious astroglial differentiation. In vivo, Zac1 and Socs3 colocalize in the neocortical ventricular zone during incipient astrogliogenesis. Zac1 overexpression in primary NSCs delays astroglial differentiation whereas knockdown of Zac1 or Socs3 facilitates formation of astroglial cells. This negative feedback loop is unrelated to Zac1's cell cycle arrest function and specific to the Jak/Stat3 pathway. Hence, reinstating Jak/Stat3 signaling in the presence of increased Zac1 expression allows for timely astroglial differentiation. Overall, we suggest that the imprinted gene Zac1 curtails astroglial differentiation of NSCs in the developing and adult brain., (Copyright © 2013 AlphaMed Press.)

The biallelic expression of the imprinted gene ZAC1/PLAGL1 underlies ≈ 60% of all cases of transient neonatal diabetes mellitus (TNDM) that present with low perinatal insulin secretion. Molecular targets of ZAC1 misexpression in pancreatic β cells are unknown. Here, we identified the guanine nucleotide exchange factor Rasgrf1 as a direct Zac1/Plagl1 target gene in murine β cells. Doubling Zac1 expression reduced Rasgrf1 expression, the stimulus-induced activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, and, ultimately, insulin secretion. Normalizing Rasgrf1 expression reversed this phenotype. Moreover, the transplantation of Zac1-overexpressing β cells failed to reinstate euglycemia in experimental diabetic mice. In contrast, Zac1 expression did not interfere with the signaling of the glucagon-like peptide 1 receptor (GLP-1R), and the GLP-1 analog liraglutide improved hyperglycemia in transplanted experimental diabetic mice. This study unravels a mechanism contributing to insufficient perinatal insulin secretion in TNDM and raises new prospects for therapy.

The zinc-finger protein Zac1 has a role as transcription factor and coregulator and plays an important role in pituitary development, maturation and tumorigenesis. Zac1 target genes control cell proliferation and hormone synthesis. While Zac1 is highly expressed in all hormone-producing cells of the pituitary, loss of expression frequently occurs in pituitary adenomas, in particular in non-functioning tumors. Zac1 lies downstream to the mitogenic MAPK and survival PI3K pathways. In turn, inhibition of the PI3K pathway by therapeutic agents, like somatostatin analogs up-regulate Zac1 expression. In fact Zac1 is an essential mediator of the antiproliferative effects of this treatment and correlates to successful outcome in acromegalic patients., (2010 Elsevier Ireland Ltd. All rights reserved.)

Objectives: ZAC1 (zinc finger protein regulating apoptosis and cell cycle arrest) is a member of the new subfamily of zinc-finger transcription factors, designated as PLAG (pleomorphic adenoma gene) family. The ZAC1 gene is maternally imprinted and is linked to developmental disorders such as growth retardation and transient neonatal diabetes mellitus. We wanted to assess whether the genetic variability of the ZAC1 gene was associated with anthropometric (weight, BMI, waist-to-hip ratio) or biochemical (plasma lipid, insulin, glucose levels, blood pressure level) phenotypes., Methods: We selected 37 independent SNPs (single nucleotide polymorphisms) or tagSNPs in the ZAC1 locus from the literature and several databases and, based on the linkage disequilibrium map, identified 27 independent SNPs. Those 27 SNPs were genotyped in a French population-based sample (n = 1155). Associations with a P value lower than 0.0019 (Bonferroni correction) were considered significant., Results: We found that women carrying the T allele of rs9403542 had lower waist-to-hip ratio (P = 0.0006) than women with the CC genotype. Also, men bearing the T allele of rs13218225 had lower systolic (P = 3.6 x 10(-5)) and diastolic (P = 4.1 x 10(-4)) blood pressure than GG men. As a consequence, the adjusted (for age, smoking habit, alcohol consumption, physical activity level and BMI) odds ratio (95% confidence interval) of hypertension for T allele carrier men was 0.55 [0.35-0.86], P = 0.009. We genotyped two other independent samples (MONICA Toulouse, n = 1130 and MONICA Strasbourg, n = 1048) for rs9403542 and rs13218225 but we could not confirm these associations., Conclusion: We found no evidence that polymorphisms in ZAC1 might influence anthropometric, biochemical or clinical parameters in French individuals.

The generally accepted paradigm of transcription by regulated recruitment defines sequence-specific transcription factors and coactivators as separate categories that are distinguished by their abilities to bind DNA autonomously. The C(2)H(2) zinc finger protein Zac1, with an established role in canonical DNA binding, also acts as a coactivator. Commensurate with this function, p73, which is related to p53, is here shown to recruit Zac1, together with the coactivators p300 and PCAF, to the p21(Cip1) promoter during the differentiation of embryonic stem cells into neurons. In the absence of autonomous DNA binding, Zac1's zinc fingers stabilize the association of PCAF with p300, suggesting its scaffolding function. Furthermore, Zac1 regulates the affinities of PCAF substrates as well as the catalytic activities of PCAF to induce a selective switch in favor of histone H4 acetylation and thereby the efficient transcription of p21(Cip1). These results are consistent with an authentic coactivator function of Zac1's C(2)H(2) zinc finger DNA-binding domain and suggest coactivation by sequence-specific transcription factors as a new facet of transcriptional control.

Zac is a C2H2 zinc finger protein, which regulates apoptosis and cell cycle arrest through DNA binding and transactivation. During tumorigenesis and in response to mitogenic activation, Zac gene expression is down-regulated in a methylation-sensitive manner. As yet, no target genes have been identified that could explain the potent antiproliferative function of Zac. Here, applying genome-wide expression analysis, we identify peroxisome proliferator-activated receptor gamma (PPARgamma) as a new bona fide Zac target gene, which is induced by direct Zac binding to the proximal PPARgamma1 promoter. We show that in human colon carcinoma cells, ZAC activates expression of PPARgamma target genes in a PPARgamma-dependent manner. Moreover, we show that treatment of pituitary tumor cells with octreotide, a somatostatin analogue, leads to Zac induction and subsequent Zac-dependent up-regulation of PPARgamma, which thereupon mediates part of the antiproliferative activity of Zac. Our work provides a first step toward elucidating a functional relationship between Zac and PPARgamma that could be relevant to the understanding of tumorigenesis and diabetes as well.

Zac is a C(2)H(2) zinc finger protein that regulates apoptosis and cell cycle arrest through DNA binding and transactivation. The coactivator proteins p300/CBP enhance transactivation through their histone acetyltransferase (HAT) activity by modulating chromatin structure. Here, we show that p300 increases Zac transactivation in a strictly HAT-dependent manner. Whereas the classic recruitment model proposes that coactivation simply depends on the capacity of the activator to recruit the coactivator, we demonstrate that coordinated binding of Zac zinc fingers and C terminus to p300 regulates HAT function by increasing histone and acetyl coenzyme A affinities and catalytic activity. This concerted regulation of HAT function is mediated via the KIX and CH3 domains of p300 in an interdependent manner. Interestingly, Zac zinc fingers 6 and 7 simultaneously play key roles in DNA binding and p300 regulation. Our findings demonstrate, for the first time, that C(2)H(2) zinc fingers can link DNA binding to HAT signaling and suggest a dynamic role for DNA-binding proteins in the enzymatic control of transcription.

Zac encodes a zinc finger protein that promotes apoptosis and cell cycle arrest and is maternally imprinted. Here, we show that Zac contains transactivation and repressor activities and that these transcriptional activities are differentially controlled by DNA binding. Zac transactivation mapped to two distinct domains. One of these contained multiple repeats of the peptide PLE, which behaved as an autonomous activation unit. More importantly, we identified two related high-affinity DNA-binding sites which were differentially bound by seven Zac C(2)H(2) zinc fingers. Zac bound as a monomer through zinc fingers 6 and 7 to the palindromic DNA element to confer transactivation. In contrast, binding as a monomer to one half-site of the repeat element turned Zac into a repressor. Conversely, Zac dimerization at properly spaced direct and reverse repeat elements enabled transactivation, which strictly correlated with DNA-dependent and -independent contacts of key residues within the recognition helix of zinc finger 7. The later ones support specific functional connections between Zac DNA binding and transcriptional-regulatory surfaces. Both classes of DNA elements were identified in a new Zac target gene and confirmed that the zinc fingers communicate with the transactivation function. Together, our data demonstrate a role for Zac as a transcription factor in addition to its role as coactivator for nuclear receptors and p53.

The ZAC gene encodes a new zinc-finger protein that concomitantly induces apoptosis and cell cycle arrest and localizes to chromosome 6q24-q25, a well-known hot spot related to cancer. ZAC is highly expressed in the anterior pituitary gland, and its ablation by antisense targeting promotes pituitary cell proliferation. Here we investigate ZAC status in pituitary tumors to evaluate its role in pituitary tumorigenesis. Interest ingly, a strong reduction or absence of ZAC mRNA and protein expres sion was detected in nonfunctioning pituitary adenomas, whereas in clin ically active pituitary neoplasias, the decrease in ZAC expression was variable. Loss of expression was not associated with a mutation of the ZAC gene. Our observations suggest that alternative mechanisms of gene inactivation and/or altered regulation of the ZAC gene occur in nonfunctioning pituitary adenomas.

Loss of chromosome 6q21-qter is the second most frequent loss of chromosomal material in sporadic breast neoplasms suggesting the presence of at least one tumor suppressor gene on 6q. We recently isolated a cDNA encoding a new zinc finger protein which we named ZAC according to its functional properties, namely induction of apoptosis and control of cell cycle progression. ZAC is expressed in normal mammary gland and maps to 6q24-q25, a recognized breast cancer hot spot on 6q. In the present report, we investigated the possible inactivation of ZAC in breast cancer cell lines and primary tumors. We detected no mutation in ZAC coding region in a panel of 45 breast tumors with allelic imbalance of 6q24-q25. However, a survey of eight breast cancer cell lines showed a deeply reduced (three cell lines) or complete loss of (five cell lines) ZAC expression. Treatment of three of these cell lines with the methylation-interfering agent 5-azacytidine induced ZAC re-expression. In addition, Northern blot and RNase protection assay analysis of ZAC expression in 23 unselected primary breast tumors showed a reduced expression in several samples. Together with its functional properties and chromosomal localization, these findings substantiate ZAC as a good candidate for the tumor suppressor gene on 6q24-q25.

Zac1 is a new zinc finger protein that concomitantly controls apoptosis and cell cycle arrest through separate pathways. The mouse Zac1 gene is mainly expressed in the pituitary gland and in different brain areas. In this study regional and cellular expression of Zac1 in the pituitary gland was determined by in situ hybridization. Zac1 messenger RNA was abundantly expressed in the anterior pituitary lobe compared with that in the intermediate and posterior lobes. Zac1 transcripts were found in all hormone-secreting cell types, with the highest levels in GH- and PRL-producing cells. To investigate the impact of Zac1 in pituitary cell proliferation, we ablated the endogenous Zac1 gene by antisense treatment in two murine cell types, AtT-20 and TtT/GF, that are representative of granular and agranular cell lineages, respectively. The decline in Zac1 protein levels under antisense treatment was accompanied by increased DNA synthesis in clonal corticotroph and folliculo-stellate cells, as demonstrated by enhanced [3H]thymidine incorporation (36% and 50%, respectively). Antisense oligonucleotides against Zac1 controlled cell proliferation in a dose-dependent way, and mutagenized antisense oligonucleotides were inert. Conclusively, our data provide the first evidence of a role for Zac1 in pituitary growth control.

We reported recently the cloning of the type I PACAP receptor by a functional expression cloning technique. Unexpectedly, we observed additional PACAP-positive pools that turned out to encode the wild-type form of the tumor suppressor gene p53 and the novel zinc finger protein Zac1, which regulates apoptosis and cell cycle arrest. Both Zac1 and p53 caused, under transient or stably regulated expression, induction of the type I PACAP receptor by transcriptional mechanisms. Transactivation of the type I PACAP receptor gene by Zac1 and p53 points to a subtle balance between death promoting and protective mechanisms. The control of these processes is central to various physiological conditions ranging from development to senescence, whereas dysregulation may lead to overt pathological outcomes, notably cancer, immune deficiency syndromes, and neurodegenerative disorders.

We previously reported the identification of mZac, a novel mouse zinc finger protein that shared with p53 the ability to regulate concomitantly apoptosis and cell cycle progression. We describe here the isolation, chromosomal localization, and functional in vitro characterization of its human homolog. hZAC is a widely expressed zinc finger protein that reveals transactivation and DNA-binding activity. hZAC inhibits tumor cell growth through induction of apoptotic cell death and G1 arrest. Thus hZAC, like its mouse counterpart, displays antiproliferative properties through pathways known to be central to the activity of p53. We mapped hZAC on chromosome 6q24-q25, a region frequently deleted in many solid tumors. Indeed, allelic loss at 6q24-q25 has been shown in breast and ovary cancers, melanomas, astrocytomas, and renal cell carcinomas. Furthermore, Abdollahi et al. [Abdollahi, A., Godwin, A. K., Miller, P. D., Getts, L. A., Schultz, D. C., Tagushi, T., Testa, J. R. & Hamilton, T. C. (1997) Cancer Res. 57, 2029-2034] recently isolated ZAC through its loss of expression in a surface epithelial ovary tumor model and accordingly named it Lot for "lost on transformation." In view of these observations, the functional properties we report here provide further arguments to consider hZAC as a tumor suppressor gene candidate.

The proliferation rate of a cell population reflects a balance between cell division, cell cycle arrest, differentiation and apoptosis. The regulation of these processes is central to development and tissue homeostasis, whereas dysregulation may lead to overt pathological outcomes, notably cancer and neurodegenerative disorders. We report here the cloning of a novel zinc finger protein which regulates apoptosis and cell cycle arrest and was accordingly named Zac1. In vitro Zac1 inhibited proliferation of tumor cells, as evidenced by measuring colony formation, growth rate and cloning in soft agar. In vivo Zac1 abrogated tumor formation in nude mice. The antiproliferative activity of Zac1 was due to induction of extensive apoptosis and of G1 arrest, which proceeded independently of retinoblastoma protein and of regulation of p21(WAF1/Cip1), p27Kip1, p57Kip2 and p16INK4a expression. Zac1-mediated apoptosis was unrelated to cell cycle phase and G1 arrest was independent of apoptosis, indicating separate control of apoptosis and cell cycle arrest. Zac1 is thus the first gene besides p53 which concurrently induces apoptosis and cell cycle arrest.