Repression Of Factors Research Articles

Disorders of sex development in Wolf\u2013Hirschhorn syndrome: a genotype\u2013phenotype correlation and MSX1 as candidate gene

BackgroundWolf–Hirschhorn (WHS) is a set of congenital physical anomalies and mental retardation associated with a partial deletion of the short arm of chromosome 4. To establish a genotype–phenotype correlation; we carried out a molecular cytogenetic analysis on two Tunisian WHS patients. Patient 1 was a boy of 1-year-old, presented a typical WHS phenotype while patient 2, is a boy of 2 days presented an hypospadias, a micropenis and a cryptorchidie in addition to the typical WHS phenotype. Both the array comparative genomic hybridization and fluorescence in situ hybridization techniques were used.ResultsResults of the analysis showed that patient 2 had a greater deletion size (4.8 Mb) of chromosome 4 than patient 1 (3.4 Mb). Here, we notice that the larger the deletion, the more genes are likely to be involved, and the more severe the phenotype is likely to be. If we analyze the uncommon deleted region between patient1 and patient 2 we found that the Muscle Segment Homeobox (MSX1) gene is included in this region. MSX1 is a critical transcriptional repressor factor, expressed in the ventral side of the developing anterior pituitary and implicated in gonadotrope differentiation. Msx1 acts as a negative regulatory pituitary development by repressing the gonadotropin releasing hormone (GnRH) genes during embryogenesis. We hypothesized that the deletion of MSX1 in our patient may deregulate the androgen synthesis.ConclusionBased on the MSX1 gene function, its absence might be indirectly responsible for the hypospadias phenotype by contributing to the spatiotemporal regulation of GnRH transcription during development.

WARTIME AND POST-WAR RAPE IN FRANCO'S SPAIN

AbstractBy examining the experience of rape in Spain in the 1930s and 1940s, this article explains how the Spanish Civil War and Franco's dictatorship dramatically increased the likelihood of women becoming victims of sexual assault. Contrary to what historians often assume, this phenomenon was not the result of rape being deliberately used as a ‘weapon of war’ or as a blunt method of political repression against women. The upsurge in sexual violence was a by-product of structural transformations in the wartime and dictatorial contexts, and it was the direct consequence, rather than the instrument, of the violent imposition of a fascist-inspired regime. Using archival evidence from numerous Spanish archives, the article historicizes rape in a wider cultural, legal, and social context and reveals the essential albeit ambiguous political nature of both wartime and post-war rape. The experience of rape was mostly shaped not by repression but structural factors such as ruralization and social hierarchization, demographic upheavals, exacerbation of violent masculinity models, the proliferation of weapons, and the influence of fascist and national-Catholic ideologies. Rape became an expression of the nature of power and social and gender relations in Franco's regime.

Flagellin-based electrochemical sensing layer for arsenic detection in water

Regular monitoring of arsenic concentrations in water sources is essential due to the severe health effects. Our goal was to develop a rapidly responding, sensitive and stable sensing layer for the detection of arsenic. We have designed flagellin-based arsenic binding proteins capable of forming stable filament structures with high surface binding site densities. The D3 domain of Salmonella typhimurium flagellin was replaced with an arsenic-binding peptide motif of different bacterial ArsR transcriptional repressor factors. We have shown that the fusion proteins developed retain their polymerization ability and have thermal stability similar to that of wild-type filament. The strong arsenic binding capacity of the monomeric proteins was confirmed by isothermal titration calorimetry (ITC), and dissociation constants (Kd) of a few hundred nM were obtained for all three variants. As-binding fibers were immobilized on the surface of a gold electrode and used as a working electrode in cyclic voltammetry (CV) experiments to detect inorganic arsenic near the maximum allowable concentration (MAC) level. Based on these results, it can be concluded that the stable arsenic-binding flagellin variant can be used as a rapidly responding, sensitive, but simple sensing layer in a field device for the MAC-level detection of arsenic in natural waters.

Tracheal Separation is Driven by NKX2-1-Mediated Repression of <i>Efnb2</i> and Regulation of Endodermal Cell Sorting

The mechanisms coupling fate specification of tissues to their physical separation remain largely unknown. The mammalian trachea and esophagus differentiate from a single tube of definitive endoderm, requiring the mutually repressive transcription factors SOX2 and NKX2-1, but how the dorsoventral site of tissue separation is defined to properly allocate tracheal and esophageal cell types is unknown. Here, we show that the EPH/EPHRIN signaling gene Efnb2 regulates tracheoesophageal separation by controlling the dorsoventral allocation of tracheal-fated cells. Ventral loss of NKX2-1 results in disruption of separation and an expansion of Efnb2 expression in the trachea that is independent of SOX2. Through chromatin immunoprecipitation, we find that NKX2-1 binds the Efnb2 gene, indicating that repression is likely direct. Finally, by inducing mosaicism for Nkx2-1 within the embryonic foregut, we generate ectopic NKX2-1/EPHRIN-B2 boundaries which organize ectopic trachea separation. Together these data demonstrate that NKX2-1 connects tracheal specification with tissue separation through the regulation of EPHRIN-B2 and tracheoesophageal cell sorting.

Identification of unrecognized host factors promoting HIV-1 latency.

To counter HIV latency, it is important to develop a better understanding of the full range of host factors promoting latency. Their identification could suggest new strategies to reactivate latent proviruses and subsequently kill the host cells (“shock and kill”), or to permanently silence these latent proviruses (“block and lock”). We recently developed a screening strategy termed “Reiterative Enrichment and Authentication of CRISPRi Targets” (REACT) that can unambiguously identify host genes promoting HIV latency, even in the presence of high background “noise” produced by the stochastic nature of HIV reactivation. After applying this strategy in four cell lines displaying different levels of HIV inducibility, we identified FTSJ3, TMEM178A, NICN1 and the Integrator Complex as host genes promoting HIV latency. shRNA knockdown of these four repressive factors significantly enhances HIV expression in primary CD4 T cells, and active HIV infection is preferentially found in cells expressing lower levels of these four factors. Mechanistically, we found that downregulation of these newly identified host inhibitors stimulates different stages of RNA Polymerase II-mediated transcription of HIV-1. The identification and validation of these new host inhibitors provide insight into the novel mechanisms that maintain HIV latency even when cells are activated and undergo cell division.

ZNF354C is a transcriptional repressor that inhibits endothelial angiogenic sprouting

Zinc finger proteins (ZNF) are a large group of transcription factors with diverse functions. We recently discovered that endothelial cells harbour a specific mechanism to limit the action of ZNF354C, whose function in endothelial cells is unknown. Given that ZNF354C has so far only been studied in bone and tumour, its function was determined in endothelial cells. ZNF354C is expressed in vascular cells and localises to the nucleus and cytoplasm. Overexpression of ZNF354C in human endothelial cells results in a marked inhibition of endothelial sprouting. RNA-sequencing of human microvascular endothelial cells with and without overexpression of ZNF354C revealed that the protein is a potent transcriptional repressor. ZNF354C contains an active KRAB domain which mediates this suppression as shown by mutagenesis analysis. ZNF354C interacts with dsDNA, TRIM28 and histones, as observed by proximity ligation and immunoprecipitation. Moreover, chromatin immunoprecipitation revealed that the ZNF binds to specific endothelial-relevant target-gene promoters. ZNF354C suppresses these genes as shown by CRISPR/Cas knockout and RNAi. Inhibition of endothelial sprouting by ZNF354C is dependent on the amino acids DV and MLE of the KRAB domain. These results demonstrate that ZNF354C is a repressive transcription factor which acts through a KRAB domain to inhibit endothelial angiogenic sprouting.

Regulation and Roles of the Nucleolus in Embryonic Stem Cells: From Ribosome Biogenesis to Genome Organization.

The nucleolus is the largest compartment of the eukaryotic cell's nucleus. It acts as a ribosome factory, thereby sustaining the translation machinery. The nucleolus is also the subnuclear compartment with the highest transcriptional activity in the cell, where hundreds of ribosomal RNA (rRNA) genes transcribe the overwhelming majority of RNAs. The structure and composition of the nucleolus change according to the developmental state. For instance, in embryonic stem cells (ESCs), rRNA genes display a hyperactive transcriptional state and open chromatin structure compared with differentiated cells. Increasing evidence indicates that the role of the nucleolus and rRNA genes might go beyond the control of ribosome biogenesis. One such role is linked to the genome architecture, since repressive domains are often located close to the nucleolus. This review highlights recent findings describing how the nucleolus is regulated in ESCs and its role in regulating ribosome biogenesis and genome organization for the maintenance of stem cell identity.

Control of Immediate Early Gene Expression for Human Cytomegalovirus Reactivation.

Human cytomegalovirus (HCMV) is a beta herpesvirus that persists for life in the majority of the world's population. The persistence of HCMV in the human population is due to the exquisite ability of herpesviruses to establish a latent infection that evades elimination by the host immune response. How the virus moves into and out of the latent state has been an intense area of research focus and debate. The prevailing paradigm is that the major immediate early promoter (MIEP), which drives robust expression of the major immediate early (MIE) transactivators, is epigenetically silenced during the establishment of latency, and must be reactivated for the virus to exit latency and re-enter productive replication. While it is clear that the MIEP is silenced by the association of repressive chromatin remodeling factors and histone marks, the mechanisms by which HCMV de-represses MIE gene expression for reactivation are less well understood. We have identified alternative promoter elements within the MIE locus that drive a second or delayed phase of MIE gene expression during productive infection. In the context of reactivation in THP-1 macrophages and primary CD34+ human progenitor cells, MIE transcripts are predominantly derived from initiation at these alternative promoters. Here we review the mechanisms by which alternative viral promoters might tailor the control of viral gene expression and the corresponding pattern of infection to specific cell types. Alternative promoter control of the HCMV MIE locus increases versatility in the system and allows the virus to tightly repress viral gene expression for latency but retain the ability to sense and respond to cell type-specific host cues for reactivation of replication.

Extensive SUMO Modification of Repressive Chromatin Factors Distinguishes Pluripotent from Somatic Cells

SummaryPost-translational modification by SUMO is a key regulator of cell identity. In mouse embryonic fibroblasts (MEFs), SUMO impedes reprogramming to pluripotency, while in embryonic stem cells (ESCs), it represses the emergence of totipotent-like cells, suggesting that SUMO targets distinct substrates to preserve somatic and pluripotent states. Using MS-based proteomics, we show that the composition of endogenous SUMOylomes differs dramatically between MEFs and ESCs. In MEFs, SUMO2/3 targets proteins associated with canonical SUMO functions, such as splicing, and transcriptional regulators driving somatic enhancer selection. In contrast, in ESCs, SUMO2/3 primarily modifies highly interconnected repressive chromatin complexes, thereby preventing chromatin opening and transitioning to totipotent-like states. We also characterize several SUMO-modified pluripotency factors and show that SUMOylation of Dppa2 and Dppa4 impedes the conversion to 2-cell-embryo-like states. Altogether, we propose that rewiring the repertoire of SUMO target networks is a major driver of cell fate decision during embryonic development.

Mini review: Advances in understanding regulation of cellulase enzyme in white-rot basidiomycetes

White-rot basidiomycetic fungi have gained a lot of scientific attention in recent years owing to their ability to produce cellulase enzymes that are of great importance in numerous industrial applications. This has seen a rise in number of studies seeking to comprehend both physical and molecular mechanisms that regulate the production of cellulase enzymes in these fungi. Cellulase has several applications in production of food and beverages, biofuel, biological detergents, pharmaceuticals, and deinking in paper and pulp industry. Enhanced understanding of genetic mechanisms that regulate cellulase production would have utility for optimal cellulase production in white-rot basidiomycetes using biotechnology approaches. Carbon catabolite repression and various transcriptional factors such as XlnR, Cre, Clr, Ace, and gna1 control expression of genes encoding cellobiohydrolase (CBH), endoglucanase (EGL) and β-glucosidase (BGL). In this review, we have consolidated and summarised some of recent findings on genetic regulation of cellulase with an aim of highlighting the general regulatory mechanisms that underlie cellulase expressions in white-rot fungi. This review further outlines some of important transcription factors that regulate cellulase genes, and key research gaps that may need to be addressed by future research.

Regulation of Ergosterol Biosynthesis in Saccharomyces cerevisiae.

Ergosterol is an essential component of fungal cell membranes that determines the fluidity, permeability and activity of membrane-associated proteins. Ergosterol biosynthesis is a complex and highly energy-consuming pathway that involves the participation of many enzymes. Deficiencies in sterol biosynthesis cause pleiotropic defects that limit cellular proliferation and adaptation to stress. Thereby, fungal ergosterol levels are tightly controlled by the bioavailability of particular metabolites (e.g., sterols, oxygen and iron) and environmental conditions. The regulation of ergosterol synthesis is achieved by overlapping mechanisms that include transcriptional expression, feedback inhibition of enzymes and changes in their subcellular localization. In the budding yeast Saccharomyces cerevisiae, the sterol regulatory element (SRE)-binding proteins Upc2 and Ecm22, the heme-binding protein Hap1 and the repressor factors Rox1 and Mot3 coordinate ergosterol biosynthesis (ERG) gene expression. Here, we summarize the sterol biosynthesis, transport and detoxification systems of S. cerevisiae, as well as its adaptive response to sterol depletion, low oxygen, hyperosmotic stress and iron deficiency. Because of the large number of ERG genes and the crosstalk between different environmental signals and pathways, many aspects of ergosterol regulation are still unknown. The study of sterol metabolism and its regulation is highly relevant due to its wide applications in antifungal treatments, as well as in food and pharmaceutical industries.

Variable pulmonary manifestations in Chitayat syndrome: Six additional affected individuals.

Hand hyperphalangism leading to shortened index fingers with ulnar deviation, hallux valgus, mild facial dysmorphism and respiratory compromise requiring assisted ventilation are the key features of Chitayat syndrome. This condition results from the recurrent heterozygous missense variant NM_006494.2:c.266A>G; p.(Tyr89Cys) in ERF on chromosome 19q13.2, encoding the ETS2 repressor factor (ERF) protein. The pathomechanism of Chitayat syndrome is unknown. To date, seven individuals with Chitayat syndrome and the recurrent pathogenic ERF variant have been reported in the literature. Here, we describe six additional individuals, among them only one presenting with a history of assisted ventilation, and the remaining presenting with variable pulmonary phenotypes, including one individual without any obvious pulmonary manifestations. Our findings widen the phenotype spectrum caused by the recurrent pathogenic variant in ERF, underline Chitayat syndrome as a cause of isolated skeletal malformations and therefore contribute to the improvement of diagnostic strategies in individuals with hand hyperphalangism.

Molecular adaptations of the blood–brain barrier promote stress resilience vs. depression

Preclinical and clinical studies suggest that inflammation and vascular dysfunction contribute to the pathogenesis of major depressive disorder (MDD). Chronic social stress alters blood-brain barrier (BBB) integrity through loss of tight junction protein claudin-5 (cldn5) in male mice, promoting passage of circulating proinflammatory cytokines and depression-like behaviors. This effect is prominent within the nucleus accumbens, a brain region associated with mood regulation; however, the mechanisms involved are unclear. Moreover, compensatory responses leading to proper behavioral strategies and active resilience are unknown. Here we identify active molecular changes within the BBB associated with stress resilience that might serve a protective role for the neurovasculature. We also confirm the relevance of such changes to human depression and antidepressant treatment. We show that permissive epigenetic regulation of cldn5 expression and low endothelium expression of repressive cldn5-related transcription factor foxo1 are associated with stress resilience. Region- and endothelial cell-specific whole transcriptomic analyses revealed molecular signatures associated with stress vulnerability vs. resilience. We identified proinflammatory TNFα/NFκB signaling and hdac1 as mediators of stress susceptibility. Pharmacological inhibition of stress-induced increase in hdac1 activity rescued cldn5 expression in the NAc and promoted resilience. Importantly, we confirmed changes in HDAC1 expression in the NAc of depressed patients without antidepressant treatment in line with CLDN5 loss. Conversely, many of these deleterious CLDN5-related molecular changes were reduced in postmortem NAc from antidepressant-treated subjects. These findings reinforce the importance of considering stress-induced neurovascular pathology in depression and provide therapeutic targets to treat this mood disorder and promote resilience.

The NMN Module Conducts Nodule Number Orchestra.

Legumes control nodule number through nodulation and autoregulation of nodulation (AON) pathways. Nodule Inception (NIN) is essential for rhizobial infection and nodule organogenesis in legumes. The GmNINa-miR172c-NNC1 (NMN) module, which consists of two positive regulators, GmNINa and miR172c, and a suppressor, NNC1, integrates both pathways. GmNINa activates miR172c to downregulate NNC1, leading to nodulation, while NNC1 inhibits miR172c expression, forming a negative feedback loop. GmNINa and NNC1 interact with each other and antagonistically fine-tune GmRIC1/RIC2 expression, turning AON on and off. Conversely, activation of AON inhibits GmNINa and miR172c expression, thereby reducing their inhibitory effects on NNC1 to attenuate both nodulation signaling and AON. The NMN module functions not only as an “accelerator” of the nodulation signal to promote nodulation but also as a “brake” on the signal by activating AON to orchestrate nodule number.

Su(var)2-10 and the SUMO Pathway Link piRNA-Guided Target Recognition to Chromatin Silencing.

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation ismediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that inDrosophila the small ubiquitin-like protein SUMOand the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.

GC content shapes mRNA storage and decay in human cells.

mRNA translation and decay appear often intimately linked although the rules of this interplay are poorly understood. In this study, we combined our recent P-body transcriptome with transcriptomes obtained following silencing of broadly acting mRNA decay and repression factors, and with available CLIP and related data. This revealed the central role of GC content in mRNA fate, in terms of P-body localization, mRNA translation and mRNA stability: P-bodies contain mostly AU-rich mRNAs, which have a particular codon usage associated with a low protein yield; AU-rich and GC-rich transcripts tend to follow distinct decay pathways; and the targets of sequence-specific RBPs and miRNAs are also biased in terms of GC content. Altogether, these results suggest an integrated view of post-transcriptional control in human cells where most translation regulation is dedicated to inefficiently translated AU-rich mRNAs, whereas control at the level of 5' decay applies to optimally translated GC-rich mRNAs.

Cohesin-dependent regulation of gene expression during differentiation is lost in cohesin-mutated myeloid malignancies

AbstractCohesin complex disruption alters gene expression, and cohesin mutations are common in myeloid neoplasia, suggesting a critical role in hematopoiesis. Here, we explore cohesin dynamics and regulation of hematopoietic stem cell homeostasis and differentiation. Cohesin binding increases at active regulatory elements only during erythroid differentiation. Prior binding of the repressive Ets transcription factor Etv6 predicts cohesin binding at these elements and Etv6 interacts with cohesin at chromatin. Depletion of cohesin severely impairs erythroid differentiation, particularly at Etv6-prebound loci, but augments self-renewal programs. Together with corroborative findings in acute myeloid leukemia and myelodysplastic syndrome patient samples, these data suggest cohesin-mediated alleviation of Etv6 repression is required for dynamic expression at critical erythroid genes during differentiation and how this may be perturbed in myeloid malignancies.

COMMD3:BMI1 Fusion and COMMD3 Protein Regulate C-MYC Transcription: Novel Therapeutic Target for Metastatic Prostate Cancer.

Gene rearrangement is reported to be associated to the aggressive phenotype and poor prognosis in prostate cancer. We identified a gene fusion between a transcription repressor (BMI1) and transcriptional factor (COMMD3) in human prostate cancer. We show that COMMD3:BMI1 fusion expression is significantly increased in prostate cancer disease in an order: normal tissue < primary < metastatic tumors (Mets). Although elevated TMPRSS-ERG/ETV fusion is reported in prostate cancer, we identified a subtype of Mets exhibiting low TMPRSS:ETV and high COMMD3:BMI1 We delineated the mechanism and function of COMMD3 and COMMD3:BMI1 in prostate cancer. We show that COMMD3 level is elevated in prostate cancer cell models, PDX models (adenocarcinoma, NECaP), and Mets. The analysis of TCGA/NIH/GEO clinical data showed a positive correlation between increased COMMD3 expression to the disease recurrence and poor survival in prostate cancer. We show that COMMD3 drives proliferation of normal cells and promotes migration/invasiveness of neoplastic cells. We show that COMMD3:BMI1 and COMMD3 regulate C-MYC transcription and C-MYC downstream pathway. The ChIP analysis showed that COMMD3 protein is recruited at the promoter of C-MYC gene. On the basis of these data, we investigated the relevance of COMMD3:BMI1 and COMMD3 as therapeutic targets using in vitro and xenograft mouse models. We show that siRNA-mediated targeting of COMMD3:BMI1 and COMMD3 significantly decreases (i) C-MYC expression in BRD/BET inhibitor-resistant cells, (ii) proliferation/invasion in vitro, and (iii) growth of prostate cancer cell tumors in mice. The IHC analysis of tumors confirmed the targeting of COMMD3-regulated molecular pathway under in vivo conditions. We conclude that COMMD3:BMI1 and COMMD3 are potential progression biomarkers and therapeutic targets of metastatic prostate cancer.

Characterisation of the invasive tumour niche using astrocyte-glioblastoma organoids and decellularised human brain

Abstract Glioblastoma therapeutic challenges are in considerable part due to myriad survival adaptations and mechanisms, which allow malignant cells to repurpose signalling pathways within discreet microenvironments. These Darwinian adaptations facilitate invasion into brain parenchyma and perivascular space or promote evasion from repressive factors that represent anti-cancer defence mechanisms. We hypothesised that pre-clinical modelling of glioma invasion by recapitulating early events occurring immediately after surgery at the glioblastoma invasive margin, could reveal the cross-talk between malignant cells and the surrounding healthy astrocytes, which facilitates tumour recurrence. We first generated transgenic H1-derived neural stem cells using CRISPR/Cas9-mediated knock-in of the YFP reporter gene under the control of the GFAP promoter. Reproducible ultrahigh-throughput AggreWells™ (19,200 micro-wells per 24-well plate) were used to create astrocyte-glioblastoma organoids, which we term ‘Gliomasphere Matrices’. YFP-labelled astrocytes were co-cultured with 10 treatment-naïve patient-derived cell lines isolated from the 5-aminolevulinic (5ALA)-determined glioblastoma invasive margin. Co-cultures were seeded upon on a sequentially constructed, time-of-flight secondary ion mass spectrometry (ToF-SIMS)-characterised 3D scaffold, composed of decellularised human brain extract with defined PEGDA hydrogel. YFP-astrocytes were purified from each of the 10 Gliomasphere Matrices using fluorescence-activated cell sorting (FACS) after 6- and 10-days co-culture. RNAseq profiling to address both putative astrocytic reprogramming by invasive glioblastoma cells and gene expression changes intrinsic to tumour cells will be discussed in relation to RNAseq data from patient-derived 5ALA FACS-purified glioblastoma invasive margin tissue. This novel multi-faceted model offers a unique opportunity to recapitulate early molecular cross-talk which facilitates glioblastoma recurrence and may be utilised for high-throughput drug screening.

Analysis of Whole-Exome Sequencing Data for Alzheimer Disease Stratified by APOE Genotype

Previous genome-wide association studies of common variants identified associations for Alzheimer disease (AD) loci evident only among individuals with particular APOE alleles. To identify APOE genotype-dependent associations with infrequent and rare variants using whole-exome sequencing. The discovery stage included 10 441 non-Hispanic white participants in the Alzheimer Disease Sequencing Project. Replication was sought in 2 independent, whole-exome sequencing data sets (1766 patients with AD, 2906 without AD [controls]) and a chip-based genotype imputation data set (8728 patients with AD, 9808 controls). Bioinformatics and functional analyses were conducted using clinical, cognitive, neuropathologic, whole-exome sequencing, and gene expression data obtained from a longitudinal cohort sample including 402 patients with AD and 647 controls. Data were analyzed between March 2017 and September 2018. Score, Firth, and sequence kernel association tests were used to test the association of AD risk with individual variants and genes in subgroups of APOE ε4 carriers and noncarriers. Results with P ≤ 1 × 10-5 were further evaluated in the replication data sets and combined by meta-analysis. Among 3145 patients with AD and 4213 controls lacking ε4 (mean [SD] age, 83.4 [7.6] years; 4363 [59.3.%] women), novel genome-wide significant associations were obtained in the discovery sample with rs536940594 in AC099552 (odds ratio [OR], 88.0; 95% CI, 9.08-852.0; P = 2.22 × 10-7) and rs138412600 in GPAA1 (OR, 1.78; 95% CI, 1.44-2.2; meta-P = 7.81 × 10-8). GPAA1 was also associated with expression in the brain of GPAA1 (β = -0.08; P = .03) and its repressive transcription factor, FOXG1 (β = 0.13; P = .003), and global cognition function (β = -0.53; P = .009). Significant gene-wide associations (threshold P ≤ 6.35 × 10-7) were observed for OR8G5 (P = 4.67 × 10-7), IGHV3-7 (P = 9.75 × 10-16), and SLC24A3 (P = 2.67 × 10-12) in 2377 patients with AD and 706 controls with ε4 (mean [SD] age, 75.2 [9.6] years; 1668 [54.1%] women). The study identified multiple possible novel associations for AD with individual and aggregated rare variants in groups of individuals with and without APOE ε4 alleles that reinforce known and suggest additional pathways leading to AD.