Identification of the BRD1 interaction network and its impact on mental disorder risk

BACKGROUND: The bromodomain containing 1 (BRD1) gene has been implicated with transcriptional regulation, brain development, and susceptibility to schizophrenia and bipolar disorder. To advance the understanding of BRD1 and its role in mental disorders, we characterized the protein and chromatin interactions of the BRD1 isoforms, BRD1-S and BRD1-L. METHODS: Stable human cell lines expressing epitope tagged BRD1-S and BRD1-L were generated and used as discovery systems for identifying protein and chromatin interactions. Protein-protein interactions were identified using co-immunoprecipitation followed by mass spectrometry and chromatin interactions were identified using chromatin immunoprecipitation followed by next generation sequencing. Gene expression profiles and differentially expressed genes were identified after upregulating and downregulating BRD1 expression using microarrays. The presented functional molecular data were integrated with human genomic and transcriptomic data using available GWAS, exome-sequencing datasets as well as spatiotemporal transcriptomic datasets from the human brain. RESULTS: We present several novel protein interactions of BRD1, including isoform-specific interactions as well as proteins previously implicated with mental disorders. By BRD1-S and BRD1-L chromatin immunoprecipitation followed by next generation sequencing we identified binding to promoter regions of 1540 and 823 genes, respectively, and showed correlation between BRD1-S and BRD1-L binding and regulation of gene expression. The identified BRD1 interaction network was found to be predominantly co-expressed with BRD1 mRNA in the human brain and enriched for pathways involved in gene expression and brain function. By interrogation of large datasets from genome-wide association studies, we further demonstrate that the BRD1 interaction network is enriched for schizophrenia risk. CONCLUSION: Our results show that BRD1 interacts with chromatin remodeling proteins, e.g. PBRM1, as well as histone modifiers, e.g. MYST2 and SUV420H1. We find that BRD1 primarily binds in close proximity to transcription start sites and regulates expression of numerous genes, many of which are involved with brain development and susceptibility to mental disorders. Our findings indicate that BRD1 acts as a regulatory hub in a comprehensive schizophrenia risk network which plays a role in many brain regions throughout life, implicating e.g. striatum, hippocampus, and amygdala at mid-fetal stages. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13073-016-0308-x) contains supplementary material, which is available to authorized users

Regulatory mechanisms for 3′-end alternative splicing and polyadenylation of the Glial Fibrillary Acidic Protein, GFAP, transcript

The glial fibrillary acidic protein, GFAP, forms the intermediate cytoskeleton in cells of the glial lineage. Besides the common GFAPα transcript, the GFAPε and GFAPκ transcripts are generated by alternative mRNA 3′-end processing. Here we use a GFAP minigene to characterize molecular mechanisms participating in alternative GFAP expression. Usage of a polyadenylation signal within the alternatively spliced exon 7a is essential to generate the GFAPκ and GFAPκ transcripts. The GFAPκ mRNA is distinct from GFAPε mRNA given that it also includes intron 7a. Polyadenylation at the exon 7a site is stimulated by the upstream splice site. Moreover, exon 7a splice enhancer motifs supported both exon 7a splicing and polyadenylation. SR proteins increased the usage of the exon 7a polyadenylation signal but not the exon 7a splicing, whereas the polypyrimidine tract binding (PTB) protein enhanced both exon 7a polyadenylation and exon 7a splicing. Finally, increasing transcription by the VP16 trans-activator did not affect the frequency of use of the exon 7a polyadenylation signal whereas the exon 7a splicing frequency was decreased. Our data suggest a model with the selection of the exon 7a polyadenylation site being the essential and primary event for regulating GFAP alternative processing

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.</p

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.</p

Gene expression responses to FUS, EWS, and TAF15 reduction and stress granule sequestration analyses identifies FET-protein non-redundant functions.

The FET family of proteins is composed of FUS/TLS, EWS/EWSR1, and TAF15 and possesses RNA- and DNA-binding capacities. The FET-proteins are involved in transcriptional regulation and RNA processing, and FET-gene deregulation is associated with development of cancer and protein granule formations in amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and trinucleotide repeat expansion diseases. We here describe a comparative characterization of FET-protein localization and gene regulatory functions. We show that FUS and TAF15 locate to cellular stress granules to a larger extend than EWS. FET-proteins have no major importance for stress granule formation and cellular stress responses, indicating that FET-protein stress granule association most likely is a downstream response to cellular stress. Gene expression analyses showed that the cellular response towards FUS and TAF15 reduction is relatively similar whereas EWS reduction resulted in a more unique response. The presented data support that FUS and TAF15 are more functionally related to each other, and that the FET-proteins have distinct functions in cellular signaling pathways which could have implications for the neurological disease pathogenesis

Fibrillary Acidic Protein, GFAP, transcript

Glia

Analysis of the Illumina HumanGW 6 BeadChip whole genome expression array.

<p>(<b>A</b>) Hierarchical clustering by Pvclust by of the significantly expressed genes in HEK293-cells transfected with siRNAs for the FET mRNAs (siFUS, siEWS, siTAF15, siFUS+siEWS+siFUS) or an unspecific control siRNA (control + and control +++). The control + is control sample for the individual FET siRNA transfections, and the control +++ for the siFUS+siEWS+siTAF15 sample, to ensure the comparisons of equal siRNA concentrations. P-values (%) are shown on the edges of the clustering, Approximately Unbiased (AU) p-values in red, and Bootstrap Probability (BP) values in green. Clusters with an AU p-value greater than 95% are highlighted by a red rectangle. (<b>B</b>) The distribution of differentially expressed genes (DEGs) in the siFUS (yellow), siEWS (blue), and siTAF15 (light red) transfected cells. The up-regulated number of genes is shown in red and down-regulated in green. The number of common DEGs is shown in the overlapping parts of the circles. (<b>C</b>) DEGs in the siFUS, siEWS, and siTAF15 transfected cells. The common DEGs were compared to the DEGs in the siFUS+siEWS+siTAF15 transfected cells. In total 637 DEGs are identified in the siFUS+siEWS+siTAF15 cells, of those are 512 unique genes (grey). 33 of the 319 common DEGs in siEWS transfected cells were found (black), 38 of 226 in FUS (red), 29 of 221 in TAF15 (green), 10 of 38 in FUS and EWS (yellow), 2 of 29 in EWS and TAF15 (dark blue), 5 of 22 in FUS and TAF15 (pink), and 8 of 21 in FUS and EWS and TAF15 were found (blue).</p

Immunostainings of the FET and TIA1 proteins in HEK293 cells after siRNA-mediated gene knock-down of the FET proteins together with arsenite induced stress.

<p>Control cells were transfected with an equal amount of siRNA with an unspecific sequence. (<b>A</b>) TIA1 and FUS immunostaining. The nuclei are counterstained by DAPI. (<b>B</b>) TIA1 and TAF15 immunostaining. The nuclei are counterstained by DAPI (<b>C</b>) TIA1 and EWS immunostaining. The nuclei are counterstained by DAPI.</p

Western blot of co-immunoprecipitation of the FLAG-TIA1 and FLAG-TIAR proteins shows no significant binding to FUS, EWS, or TAF15.

<p>(<b>A</b>) Co-immunoprecipitation conducted without RNase A. The used FLAG cell line is shown above the blot. “Emp” is HEK293-cells without FLAG vector used as control. Unstressed control cells are marked (-), and stressed arsenite treated cells (Ars). (*) marks an unidentified background protein, and HC and LC is heavy and light chain, respectively, from the used mouse antibodies. HuR is used as control protein for an intact RNA dependent interaction to TIA1 and TIAR. (<b>B</b>) The co-immunoprecipitation conducted with RNase A. (*) marks an unidentified background protein, and HC and LC is heavy and light chain respectively from the used mouse antibodies.</p