Whole-genome and RNA sequencing reveal variation and transcriptomic coordination in the developing human prefrontal cortex

SummaryVariation in gene expression underlies neurotypical development, while genomic variants contribute to neuropsychiatric disorders. BrainVar is a unique resource of paired whole-genome sequencing and bulk-tissue RNA-sequencing from the human dorsolateral prefrontal cortex of 176 neurotypical individuals across prenatal and postnatal development, providing the opportunity to assay genomic and transcriptomic variation in tandem. Leveraging this resource, we identified rare premature stop codons with commensurate reduced and allele-specific expression of corresponding genes, and common variants that alter gene expression (expression quantitative trait loci, eQTLs). Categorizing eQTLs by prenatal and postnatal effect, genes affected by temporally-specific eQTLs, compared to constitutive eQTLs, are enriched for haploinsufficiency, protein-protein interactions, and neuropsychiatric disorder risk loci. Expression levels of over 12,000 genes rise or fall in a concerted late-fetal transition, with the transitional genes enriched for cell type specific genes and neuropsychiatric disorder loci, underscoring the importance of cataloguing developmental trajectories in understanding cortical physiology and pathology.HighlightsWhole-genome and RNA-sequencing across human prefrontal cortex development in BrainVarGene-specific developmental trajectories characterize the late-fetal transitionIdentification of constitutive, prenatal-specific, postnatal-specific, and rare eQTLsIntegrated analysis reveals genetic and developmental influences on CNS traits and disorders

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Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6690-6701.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6690-6701

Author(s):

H Koizumi ◽

M F Horta ◽

B S Youn ◽

K C Fu ◽

B S Kwon ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Site ◽

Protein Interactions ◽

Regulatory Element ◽

Killer Cell ◽

Mobility Shift ◽

Specific Expression ◽

Native Molecular Mass ◽

Mobility Shift Assay

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

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Whole-Genome and RNA Sequencing Reveal Variation and Transcriptomic Coordination in the Developing Human Prefrontal Cortex

Cell Reports ◽

10.1016/j.celrep.2020.03.053 ◽

2020 ◽

Vol 31 (1) ◽

pp. 107489 ◽

Cited By ~ 19

Author(s):

Donna M. Werling ◽

Sirisha Pochareddy ◽

Jinmyung Choi ◽

Joon-Yong An ◽

Brooke Sheppard ◽

...

Keyword(s):

Prefrontal Cortex ◽

Rna Sequencing ◽

Whole Genome

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Whole Genome Sequencing and RNA Sequencing of 27 Patients with Persistent Polyclonal B-Cell Lymphocytosis Reveals a High Mutation Frequency/Overexpression of Lymphoma Associated Genes: Really a Benign Disorder?

Blood ◽

10.1182/blood-2018-99-111480 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 924-924

Author(s):

Anna Stengel ◽

Alexander Höllein ◽

Wolfgang Kern ◽

Manja Meggendorfer ◽

Claudia Haferlach ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Whole Genome Sequencing ◽

Rna Sequencing ◽

Genome Sequencing ◽

Splice Site ◽

Mutation Frequency ◽

Whole Genome ◽

Employment Equity ◽

Equity Ownership

Abstract Background: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare disorder, occurs almost exclusively in smoking women and is characterized by a chronic polyclonal lymphocytosis with circulating binucleated lymphocytes, clonal cytogenetic abnormalities involving chromosome 3, and chromosomal instability. Outcome of PPBL patients is mostly benign, but subsequent malignancies (non-Hodgkin´s lymphomas and solid tumors) were described. Potential molecular factors leading to their development are yet unclear. Aims: Detailed molecular genetic characterization of PPBL by whole genome sequencing (WGS) and RNA sequencing (RNAseq) in comparison to the well-characterized lymphoid malignancy CLL. Patient cohorts and methods: The total cohort comprised 27 PPBL (3 male, 24 female) and 250 CLL cases (163 male, 87 female). WGS was performed for all patients: 150bp paired-end reads where generated on Illumina HiseqX and NovaSeq 6000 machines (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. To remove potential germline variants, each variant was queried against the gnomAD database, variants with global population frequencies >1% where excluded. Final analysis was performed only on protein-altering and splice-site variants. For further analysis, a virtual panel of 355 lymphoid genes was selected. All reported p-values are two-sided and were considered significant at p<0.05. For gene expression analysis, estimated gene counts were normalized applying Trimmed mean of M-values (TMM) normalization method and the resulting log2 counts per million (CPMs) were used as a proxy of gene expression in each sample. Genes were kept if they were expressed (> 5 CPM) in at least 66% of the samples. Genes with FDR (false discovery rate) < 0.05 and an absolute logFC > 1.5 were considered differentially expressed (DE). Results: Median age was 46 years for PPBL patients (range: 23-67 years) and 67 years for CLL patients (range: 39-94 years). Mean number of mutations per patient was 18 for PPBL and 20 for CLL. For both entities, the majority of mutations were missense mutations (88% in PPBL vs. 81% in CLL), followed by splice-site mutations (7% vs. 10%), other mutation types were only rarely detected. In PPBL, 42 genes were found to be mutated at a frequency of >15%, including ATM (22%), CREBBP (19%), NCOR2 (19%), AHNAK2 (15%), JAK3 (15%), NOTCH2 (15%) and TRAF1 (15%), all of which have been associated with a variety of cancers. Moreover, ATM, NOTCH2 and TRAF1 mutations were described before to be associated with lymphomas. In PPBL patients, mutations in TRAF1 and ATM as well as mutations in TRAF1 and NOTCH2 were found to be mutually exclusive. For CLL patients, 29 genes showed a mutation frequency of >15%, comprising ATM (26%), KMT2D (23%), NOTCH1 (23%), LRP1B (19%), TP53 (16%) and CREBBP (15%). Comparison of the mutation frequencies between the two entities revealed several genes with significant differences: whereas mutations in CKAP5 (11% vs. 2%, p=0.022), DNMT3A (11% vs. 3%, p=0.033), MAP2 (19% vs. 4%, p=0.009), ROBO1 (15% vs. 4%, p=0.046) and TRAF1 (15% vs. 2%, p=0.006) were found to be more frequent in PPBL cases compared to CLL cases, KMT2D (4% vs. 23%, p=0.014), TDRD6 (0% vs. 14%, p=0.032) and TP53 (4% vs. 16%, p=0.048) mutations were more abundantly detected in CLL patients. Moreover, NOTCH1 was mutated more frequently in CLL cases (7% vs. 23%, p=0.082), whereas mutated NOTCH2 (known to be frequently mutated in splenic marginal zone lymphoma), was more abundant in PPBL patients (15% vs. 6%, p=0.116), although both correlations were not statistically significant. Gene expression analyses by RNAseq revealed 337 genes to be differentially expressed between the entities. 207 genes were upregulated in PPBL, including PTPRK, CXCR1, BCL11B, CEPBA, CCR4 and MYC, whereas 130 genes were found to be upregulated in CLL cases, comprising ID3, BCL2, FGF2 and FLT1. Conclusions: 1) WGS analysis identifies high frequencies of cancer/lymphoma-associated gene mutations in PPBL, including mutated ATM, NOTCH2 and TRAF1. 2) Five genes showed a higher mutation frequency compared to CLL including TRAF1,DNMT3A, CKAP5 and MAP2. 3) Lymphoma associated genes (BCL11B and MYC) were overexpressed in PPBL vs CLL. 4) Taken together our results question PPBL as a benign entity and identify molecular markers that might contribute to development of subsequent malignancies. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Single-cell transcriptomics predicts relapse in MLL-rearranged acute lymphoblastic leukemia in infants

10.1101/2020.04.14.20056580 ◽

2020 ◽

Author(s):

Tito Candelli ◽

Pauline Schneider ◽

Patricia Garrido Castro ◽

Luke A. Jones ◽

Rob Pieters ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

Risk Stratification ◽

Single Cell ◽

Rna Sequencing ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

List Type ◽

Relapse Risk ◽

Single Cell Rna Sequencing

AbstractInfants with MLL-rearranged acute lymphoblastic leukemia (ALL) undergo intense therapy to counter a highly aggressive leukemia with survival rates of only 30-40%. The majority of patients initially show therapy response, but in two-thirds of cases the leukemia returns, typically during treatment. Accurate relapse prediction would enable treatment strategies that take relapse risk into account, with potential benefits for all patients. Through analysis of diagnostic bone marrow biopsies, we show that single-cell RNA sequencing can predict future relapse occurrence. By analysing gene modules derived from an independent study of the gene expression response to the key drug prednisone, individual leukemic cells are predicted to be either resistant or sensitive to treatment. Quantification of the proportion of cells classified by single-cell transcriptomics as resistant or sensitive, accurately predicts the occurrence of future relapse in individual patients. Strikingly, the single-cell based classification is even consistent with the order of relapse timing. These results lay the foundation for risk-based treatment of MLL-rearranged infant ALL, through single-cell classification. This work also sheds light on the subpopulation of cells from which leukemic relapse arises. Leukemic cells associated with high relapse risk are characterized by a smaller size and a quiescent gene expression program. These cells have significantly fewer transcripts, thereby also demonstrating why single-cell analyses may outperform bulk mRNA studies for risk stratification. This study indicates that single-cell RNA sequencing will be a valuable tool for risk stratification of MLL-rearranged infant ALL, and shows how clinically relevant information can be derived from single-cell genomics.Key PointsSingle-cell RNA sequencing accurately predicts relapse in MLL-rearranged infant ALLIdentification of cells from which MLL-rearranged infant ALL relapses occur

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Dynamics of gene expression in single root cells ofA. thaliana

10.1101/448514 ◽

2018 ◽

Cited By ~ 3

Author(s):

Ken Jean-Baptiste ◽

José L. McFaline-Figueroa ◽

Cristina M. Alexandre ◽

Michael W. Dorrity ◽

Lauren Saunders ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Developmental Trajectories ◽

Cell Types ◽

Cell Type ◽

Specific Expression ◽

Root Cells ◽

Cell Lineages ◽

Cell Type Specific Expression ◽

Cell Type Specific

ABSTRACTSingle-cell RNA-seq can yield high-resolution cell-type-specific expression signatures that reveal new cell types and the developmental trajectories of cell lineages. Here, we apply this approach toA. thalianaroot cells to capture gene expression in 3,121 root cells. We analyze these data with Monocle 3, which orders single cell transcriptomes in an unsupervised manner and uses machine learning to reconstruct single-cell developmental trajectories along pseudotime. We identify hundreds of genes with cell-type-specific expression, with pseudotime analysis of several cell lineages revealing both known and novel genes that are expressed along a developmental trajectory. We identify transcription factor motifs that are enriched in early and late cells, together with the corresponding candidate transcription factors that likely drive the observed expression patterns. We assess and interpret changes in total RNA expression along developmental trajectories and show that trajectory branch points mark developmental decisions. Finally, by applying heat stress to whole seedlings, we address the longstanding question of possible heterogeneity among cell types in the response to an abiotic stress. Although the response of canonical heat shock genes dominates expression across cell types, subtle but significant differences in other genes can be detected among cell types. Taken together, our results demonstrate that single-cell transcriptomics holds promise for studying plant development and plant physiology with unprecedented resolution.

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The IGLV3-21R110 Defines a Subset of Chronic Lymphocytic Leukemia with Intermediate Epigenetic Subtype and Poor Outcome

Blood ◽

10.1182/blood-2020-134786 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 43-44

Author(s):

Ferran Nadeu ◽

Romina Royo ◽

Guillem Clot ◽

Martí Duran-Ferrer ◽

Alba Navarro ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Rna Sequencing ◽

Prognostic Value ◽

Lymphocytic Leukemia ◽

Whole Genome ◽

Biological Features ◽

Mutational Status ◽

Bcr Signaling

Introduction: B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B-cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling conferring aggressive behavior. Epigenetic studies have defined three CLL subtypes based on methylation signatures reminiscent of pre- and post-germinal center B-cells named naïve-like (n-CLL), intermediate (i-CLL) and memory-like CLL (m-CLL) with different biological features. i-CLL carry a borderline IGHV mutational load and a significant higher usage of IGHV3-21/IGLV3-21. The integration of these factors might translate into novel insights in CLL pathogenesis with implications on the proposed stratification of the patients. Aim: To determine the clinical and biological features of the IGLV3-21R110 in CLL in the light of the epigenetic subtypes and immunogenetic, genomic and transcriptomic landscapes of the tumors. Methods: We characterized the immunoglobulin (IG) gene of 584 CLL cases from whole-genome/exome and RNA sequencing using our recently developed algorithm IgCaller (Nadeu et al., Nat. Commun. 2020) and MiXCR, respectively. The genomic makeup of the tumors was obtained from whole-genome/exome sequencing while RNA sequencing data for 369 cases was used for gene expression analyses. Expression levels of WNT5A and WNT5B were verified by quantitative PCR with reverse transcriptase. Primary end points were time to first treatment (TTFT) and overall survival (OS) calculated from the date of diagnosis. All patients gave written informed consent. The study was approved by the Ethics Committee of the Hospital Clínic of Barcelona. Results: The IGLV3-21R110 was detected in 6.5% of cases being similarly distributed between mutated (6.5%) and unmutated (6.6%) IGHV cases (P=0.56). In contrast, the IGLV3-21R110 was found in 30/79 (38%) i-CLL compared to only 5/291 (1.7%) m-CLL and 1/189 (0.5%) n-CLL (P<0.001). All stereotyped subset #2 cases carried IGLV3-21R110 while 62% of IGLV3-21R110 i-CLL had non-stereotyped IG genes. IGLV3-21R110 i-CLL had a borderline IGHV mutational status (median 97.7%) that was higher than i-CLL lacking the IGLV3-21R110 (median 96.2%, P=0.005). IGLV3-21R110 i-CLL had significantly higher number of SF3B1 and ATM mutations, and total number of driver alterations. Nonetheless, the R110 mutation was the sole alteration in one i-CLL case and accompanied only by del(13q) in three. Although composite regarding IGHV mutational status, IGLV3-21R110 i-CLL transcriptomically resembled naïve-like/unmutated IGHV CLL and had a specific expression signature of 64 genes with overexpression of WNT5A and WNT5B as hallmarks. No differences were observed in the expression profile of subset #2 and non-subset #2 IGLV3-21R110 i-CLL tumors. On the other hand, i-CLL lacking the IGLV3-21R110 phenotypically mirrored memory-like/mutated IGHV cases. In relation to prognosis, IGLV3-21R110 i-CLL had a short TTFT and OS similar to n-CLL/unmutated IGHV cases whereas non-IGLV3-21R110 i-CLL had a good prognosis similar to memory-like/mutated IGHV. Therefore, i-CLL cases, which have been associated with an intermediate prognosis between m-CLL and n-CLL in previous studies, can be divided in two subgroups of cases with opposed clinical evolutions based on the IGLV3-21R110. Indeed, the IGLV3-21R110 and n-CLL subtype retained independent prognostic value in multivariate analyses while the i-CLL lost its prognostic prediction both for TTFT and OS. The prognostic value of the IGLV3-21R110 was also independent of the IGHV mutational status. In terms of applicability in the clinics, all n-CLL cases were classified as unmutated IGHV and 98% of m-CLL were mutated IGHV. Thus, either a complete IG characterization (IGHV mutational status and IGLV3-21R110) or the integration of the n-CLL subtype and IGLV3-21R110 identified virtually the same subset of patients with aggressive disease. Conclusions: The IGLV3-21R110 defines a CLL subset with borderline IGHV mutations, specific driver alterations, a gene expression signature including WNT5A/B overexpression, and an unfavorable prognosis independent of the IGHV mutational status and epigenetic subtypes. Our findings support the identification of IGLV3-21R110 CLL as a particular subgroup of the disease with relevance in the risk stratification of the patients. Disclosures Nadeu: Janssen: Honoraria. Campo:NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America..

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Intra- and intergenerational changes in the cortical DNA methylome in response to therapeutic intermittent hypoxia in mice

Physiological Genomics ◽

10.1152/physiolgenomics.00094.2019 ◽

2020 ◽

Vol 52 (1) ◽

pp. 20-34 ◽

Cited By ~ 1

Author(s):

Krystal Courtney D. Belmonte ◽

Jarrod C. Harman ◽

Nicholas A. Lanson ◽

Jeffrey M. Gidday

Keyword(s):

Gene Expression ◽

Prefrontal Cortex ◽

Protein Interactions ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Protein Protein Interactions ◽

Dna Methylome ◽

Methylated Dna ◽

Modified Dna ◽

F1 Generation

Recent evidence from our laboratory documents functional resilience to retinal ischemic injury in untreated mice derived from parents exposed to repetitive hypoxic conditioning (RHC) before breeding. To begin to understand the epigenetic basis of this intergenerational protection, we used methylated DNA immunoprecipitation and sequencing to identify genes with differentially methylated promoters (DMGPs) in the prefrontal cortex of mice treated directly with the same RHC stimulus (F0-RHC) and in the prefrontal cortex of their untreated F1-generation offspring (F1-*RHC). Subsequent bioinformatic analyses provided key mechanistic insights into how changes in gene expression secondary to promoter hypo- and hypermethylation might afford such protection within and across generations. We found extensive changes in DNA methylation in both generations consistent with the expression of many survival-promoting genes, with twice the number of DMGPs in the cortex of F1*RHC mice relative to their F0 parents that were directly exposed to RHC. In contrast to our hypothesis that similar epigenetic modifications would be realized in the cortices of both F0-RHC and F1-*RHC mice, we instead found relatively few DMGPs common to both generations; in fact, each generation manifested expected injury resilience via distinctly unique gene expression profiles. Whereas in the cortex of F0-RHC mice, predicted protein-protein interactions reflected activation of an anti-ischemic phenotype, networks activated in F1-*RHC cortex comprised networks indicative of a much broader cytoprotective phenotype. Altogether, our results suggest that the intergenerational transfer of an acquired phenotype to offspring does not necessarily require the faithful recapitulation of the conditioning-modified DNA methylome of the parent.

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Integrated Analysis Reveals hsa-miR-142 as a Representative of a Lymphocyte-Specific Gene Expression and Methylation Signature

Cancer Informatics ◽

10.4137/cin.s9037 ◽

2012 ◽

Vol 11 ◽

pp. CIN.S9037 ◽

Cited By ~ 14

Author(s):

Bill Andreopoulos ◽

Dimitris Anastassiou

Keyword(s):

Gene Expression ◽

Cell Adhesion ◽

The Cancer Genome Atlas ◽

Integrated Analysis ◽

Specific Gene ◽

Data Types ◽

Multiple Cancer ◽

Specific Expression ◽

Cancer Types ◽

Cell Cell

Gene expression profiling has provided insights into different cancer types and revealed tissue-specific expression signatures. Alterations in microRNA expression contribute to the pathogenesis of many types of human diseases. Few studies have integrated all levels of gene expression, miRNA and methylation to uncover correlations between these data types. We performed an integrated profiling to discover instances of miRNAs associated with a gene expression and DNA methylation signature across multiple cancer types. Using data from The Cancer Genome Atlas (TCGA), we revealed a concordant gene expression and methylation signature associated with the microRNA hsa-miR-142 across the same samples. In all cancer types examined, we found a signature of co-expression of a gene set R and methylated sites M, which correlate positively (M+) or negatively (M–) with the expression of hsa-miR-142. The set R consistently contains many genes, such as TRAF3IP3, NCKAP1L, CD53, LAPTM5, PTPRC, EVI2B, DOCK2, LCP2, CYBB and FYB. The signature is preserved across glioblastoma, ovarian, breast, colon, kidney, lung, uterine and rectum cancer. There is 28% overlap of methylation sites in M between glioblastoma (GBM) and ovarian cancer. There is 60% overlap of genes in R between GBM and ovarian ( P = 1.3e−-11). Most of the genes in R are known to be expressed in lymphocytes and haematopoietic stem cells, while M reflects membrane proteins involved in cell-cell adhesion functions. We speculate that the hsa-miR-142 associated signature may signal haematopoietic-specific processes and an accumulation of methylation events triggering a progressive loss of cell-cell adhesion. We also observed that GBM samples belonging to the proneural subtype tend to have underexpressed hsa-miR-142 and R genes, hypomethylated M+ and hypermethylated M–, while the mesenchymal samples have the opposite profile.

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DNA elements with AT-rich core sequences direct pituitary cell-specific expression of the pro-opiomelanocortin gene in transgenic mice

Biochemical Journal ◽

10.1042/bj3120827 ◽

1995 ◽

Vol 312 (3) ◽

pp. 827-832 ◽

Cited By ~ 20

Author(s):

B Liu ◽

M Mortrud ◽

M J Low

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Protein Interactions ◽

Transcriptional Activator ◽

Pituitary Tumour ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Specific Gene ◽

Specific Expression ◽

Pomc Gene

Corticotrophs are the first fully differentiated cells to appear in the anterior pituitary during organogenesis and are distinguished by pro-opiomelanocortin (POMC) gene expression. Earlier studies in our laboratory defined three DNA regions (sites 1, 2 and 3) within promoter sequences at the 5′-end of the rat POMC gene (-323/-34) that cooperatively targeted cell-specific gene expression to corticotrophs and melanotrophs in transgenic mice. In this study we analysed the DNA-nuclear protein interactions underlying this functional activity. We demonstrated that the transcriptional activator SP1 interacts with GC-rich regions in sites 1 (-146/-136) and 2 (-201/-192) and an unidentified protein, which we call PP1 (putative pituitary POMC1), interacts with AT-rich regions in sites 2 (-202/-193) and 3 (-262/-253). The PP1-binding activity appears to be specific to cells that express the POMC gene because it was detected in nuclear extracts prepared from AtT20 corticotroph cells and mouse melanotroph tumours but not from GH4 pituitary tumour cells, HeLa cells or liver. Site-directed mutagenesis of core binding sequences demonstrated that PP1 is required for the correct cell-specific expression of the POMC gene in the pituitary gland of transgenic mice and SP1 appears to support such an expression. The best core binding sequence for PP1 is TAAT, a possible transcription factor homeodomain contact site. However, PP1 is distinct from Brn 3.0, a POU protein that also binds to site 3. We conclude that PP1 is a transcriptional activator for pituitary-specific POMC gene expression.

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