Alternative Transcriptional Regulation Research Articles

SURF: integrative analysis of a compendium of RNA-seq and CLIP-seq datasets highlights complex governing of alternative transcriptional regulation by RNA-binding proteins

Advances in high-throughput profiling of RNA-binding proteins (RBPs) have resulted inCLIP-seq datasets coupled with transcriptome profiling by RNA-seq. However, analysis methods that integrate both types of data are lacking. We describe SURF, Statistical Utility for RBP Functions, for integrative analysis of large collections of CLIP-seq and RNA-seq data. We demonstrate SURF’s ability to accurately detect differential alternative transcriptional regulation events and associate them to local protein-RNA interactions. We apply SURF to ENCODE RBP compendium and carry out downstream analysis with additional reference datasets. The results of this application are browsable at http://www.statlab.wisc.edu/shiny/surf/.

The regulation of PTC containing transcripts of the human NDUFS4 gene of complex I of respiratory chain and the impact of pathological mutations

The regulation of alternative transcripts of the NDUFS4 gene of complex I of the respiratory chain has been studied in human cell lines. One of the alternative transcripts (SV1) is subjected to the NMD degradation pathway which involves the hUPF1 and hUPF2 factors. Another transcript (SV3) appears to be controlled in the nuclear fraction and to be enhanced when hUPF1 is depleted, but unaffected by translation inhibitors or when hUPF2 expression is down-regulated. A pathological homozygous nonsense mutation in exon 1, found in a patient affected by mitochondrial disorder, inactivated in the patient's fibroblasts NMD degradation of SV1 and enhanced the nuclear production of SV3. In another patient with a homozygous splice acceptor site mutation in intron 1, SV3, which was the only transcript of NDUFS4 gene to be produced, accumulated in fibroblasts.

Common Transcriptional Control Complexes of Murine CD21 and CD23 Suggests Stage Specific Repression (35.35)

Abstract The expression of CD21 and CD23 is coincident in differentiating B cells during the T1 to T2 splenic transition stage. To define the factors regulating the expression of CD21 and CD23, we have used Chromatin Immunoprecipitation to map candidate transcription factors. Total naïve splenocytes showed constitutive binding of Oct1, NFATc3, YY1, NFkB-p52, PU.1, Pax5, E2A and RBP-Jk; to CD21 sequences (promoter and first intron) and NFkB-p52, Pax5, NFATc3, E2A and RBP-Jk to CD23 promoter sequences. Analysis of naïve T and B cell subsets showed constitutive binding of YY1, NFkB-p52, Pax5, Oct1 and NFATc3 proteins to CD21 sequences and NFkB-p52, Pax5, NFATc3 to CD23 promoter in B cells, but no specific binding of NFATc3 or Pax5 in T cell populations. Transcripts and protein for various NFAT species are present in splenic B and T cell (NFATc1, c2, c3, c4, NFAT5), but only a subset of NFAT proteins were defined to form constitutive complexes with the CD21 and CD23 gene sequences of resting T and B cells. The shared transcriptional control elements of CD21 and CD23 did not suggest an obvious means of alternative transcriptional regulation of these genes in marginal zone (MZ) B cells where CD21 cell surface expression and transcription are high and that of CD23 is absent or very low. These data suggest a model where MZ cells are derived directly from T1 splenic precursors. NIH-AI-24158 to JHW and NIH-AI-32223 to JJW

Monocytes, but not macrophages, produce the eosinophil cationic protein.

The eosinophil cationic protein (ECP) is a cytotoxic protein with ribonuclease activity, produced and stored in bone marrow eosinophil myelocytes. Mature circulating eosinophils contain about 10 pg ECP per cell. The aim of this study was to investigate the possibility that monocytes produce and store ECP. By results from flow cytometry and specific protein measurement it is shown that human monocytes contain ECP (monocytes about 10 fg ECP per cell). RT-PCR analysis indicated the presence of mRNA coding for ECP in blood monocytes but not in alveolar macrophages. Furthermore, mRNA coding for ECP and low amounts of the protein were found in three myeloid cell lines representing different stages of monocytic differentiation. Differentiation of U-937 cells to macrophages induced lowered transcription of the ECP gene and reduced protein production. Immunohistochemical staining of lung tissue indicated that lung macrophages do not contain ECP. It is concluded that ECP is produced to a low extent by human monocytes and that the production is shut down during macrophage differentiation. This might indicate an alternative transcriptional regulation of the ECP gene in the monocytic lineage compared to the eosinophil lineage.

Monocytes, but not macrophages, produce the eosinophil cationic protein

The eosinophil cationic protein (ECP) is a cytotoxic protein with ribonuclease activity, produced and stored in bone marrow eosinophil myelocytes. Mature circulating eosinophils contain about 10 pg ECP per cell. The aim of this study was to investigate the possibility that monocytes produce and store ECP. By results from flow cytometry and specific protein measurement it is shown that human monocytes contain ECP (monocytes about 10 fg ECP per cell). RT‐PCR analysis indicated the presence of mRNA coding for ECP in blood monocytes but not in alveolar macrophages. Furthermore, mRNA coding for ECP and low amounts of the protein were found in three myeloid cell lines representing different stages of monocytic differentiation. Differentiation of U‐937 cells to macrophages induced lowered transcription of the ECP gene and reduced protein production. Immunohistochemical staining of lung tissue indicated that lung macrophages do not contain ECP. It is concluded that ECP is produced to a low extent by human monocytes and that the production is shut down during macrophage differentiation. This might indicate an alternative transcriptional regulation of the ECP gene in the monocytic lineage compared to the eosinophil lineage.

Evolution and diversification of the nuclear receptor superfamily.

Nuclear receptors play an important part in the regulation of cell growth and differentiation by providing a direct link between signaling molecules and the transcriptional response.1 These receptors are grouped into a large superfamily which includes receptors for steroid hormones, vitamin D, ecdysone, retinoic acids (alltrans or 9-cis isoforms), or thyroid hormones. It was recently discovered that fatty acids, farnesol, or prostaglandin J2 metabolites may act through binding to nuclear receptors. In addition to these receptors for known ligands, an increasing number of “orphan” receptors have been described. It is not yet known if all of these orphan receptors indeed have a ligand still to be identified, if they act in a constitutive manner, or if they have an alternative transcriptional regulation mechanism. To date, the family of nuclear receptors contains more than 60 members.1 The modular organization of nuclear receptors, the various degrees of conservation between their respective domains, and the importance of nuclear receptors for many physiological processes in both arthropods and vertebrates have led several authors to study these molecules from an evolutionary point of view. Molecular phylogeny studies have led to a classification of the family into three subfamilies: (1) a large subfamily containing TR, RAR, PPAR, VDR, and EcR; (2) a subfamily containing RXR, HNF4, and COUP-TF; and finally (3) a subfamily containing the steroid receptors with the orphan receptors ERR1 and ERR2.2 This classification was confirmed by other studies using only the C domain.3,4 Nevertheless, the number of characterized receptors has greatly increased in the last few years, and the power of resolution of molecular phylogeny methods has been improved. In this paper we present briefly a new molecular phylogeny of the nuclear receptor superfamily. Furthermore, we tested the validity of this phylogenetic model by identifying homologs of known nuclear receptors in early metazoans.

Transcription of the vasoactive intestinal peptide gene in response to glucocorticoids: differential regulation of alternative transcripts is modulated by a labile protein in rat anterior pituitary

Expression of the vasoactive intestinal peptide (VIP) gene is controled by glucocorticoids in a tissue- and endocrine status-specific manner. We have investigated the molecular mechanisms that determine glucocorticoid regulation of VIP gene expression in the rat pituitary. In initial experiments, using explant cultures of rat pituitary glands, we have demonstrated that treatment with the glucocorticoid agonist dexamethasone leads to a marked increase in VIP mRNA levels. This effect was found to be selective for the larger of two alternatively polyadenylated VIP transcripts, and in addition, protein synthesis inhibitors markedly enhanced the magnitude of this response indicating that a labile pituitary protein acts to attenuate the transcript-selective response to glucocorticoids. Nuclear run-on analysis of transcription demonstrated that the effects of dexamethasone in vitro are mediated largely, if not completely, at the level of transcription. In order to investigate the role of VIP promoter sequence in the glucocorticoid response, we then demonstrated that the activity of rat VIP gene promoter/reporter constructs in GH3 pituitary cells are up-regulated by dexamethasone. This up-regulation is virtually abolished following removal of promoter sequence between −162 and −89 of the start of transcription. Using an in vitro electrophoretic mobility shift assay, we have also demonstrated that this region of the promoter binds recombinant glucocorticoid receptor protein. The results of our study therefore indicate a direct mechanism of action for the modulation of VIP gene expression by glucocorticoids, and furthermore provide evidence of a mechanism that permits selective glucocorticoid regulation of alternative VIP transcripts.

Intronic sequence with both negative and positive effects on the regulation of alternative transcripts of the chicken beta tropomyosin transcripts.

The chicken beta tropomyosin gene generates three major transcripts by alternative splicing. A pair of internal exons are spliced in a mutually exclusive manner and their utilisation is developmentally regulated. Exon 6A and exon 6B are used respectively in myoblasts and myotubes during the process of differentiation of muscle cells. We have previously reported that, in myoblasts, exon 6B is skipped because of a negative regulation which involves intron as well as exon sequences. In this report, we describe a previously uncharacterized intronic element which is involved in the regulation of the splicing of both exons 6A and 6B. This cis-element is localized 37nt downstream of exon 6A and is approximately 30nt long. Its deletion, as well as modification of its sequence, results in the activation of the use of exon 6B and, at the same time, in the inhibition of the use of exon 6A. The mechanisms by which this region could act are further discussed.