Summary Recent genome-wide studies have demonstrated pausing of RNA polymerase II (Pol II) occurred on many vertebrate genes. By genetic studies in the zebrafish tif1γ mutant moonshine we found that loss of function of Pol II-associated factors PAF or DSIF rescued erythroid gene transcription in tif1γ-deficient animals. Biochemical analysis established physical interactions among TIF1γ, the blood-specific SCL transcription complex, and the positive elongation factors p-TEFb and FACT. ChIP assays in human CD34+ cells supported a TIF1γ-dependent recruitment of positive elongation factors to erythroid genes to promote transcription elongation by counteracting Pol II pausing. Our study establishes a mechanism for regulating tissue cell fate and differentiation through transcription elongation.
Promoter-proximal pausing by RNA polymerase II (Pol II) is a well-established mechanism to control the timing, rate, and possibly the magnitude of transcriptional responses. Recent studies have shown that cellular signaling pathways can regulate gene transcription and signaling outcomes by controlling Pol II pausing in a wide array of biological systems. Identification of the proteins and small molecules that affect the establishment and release of paused Pol II is shedding new light on the mechanisms and biology of Pol II pausing. This review will focus on the interplay between cellular signaling pathways and Pol II pausing during normal development and under disease conditions.
MSL complexes bind the single male X chromosome in Drosophila to increase transcription approximately 2-fold. Complexes contain at least five proteins and two noncoding RNAs, roX1 and roX2. The mechanism of X chromosome binding is not known. Here, we identify a 110 bp sequence in roX2 characterized by high-affinity MSL binding, male-specific DNase I hypersensitivity, a shared consensus with the otherwise dissimilar roX1 gene, and conservation across species. Mutagenesis of evolutionarily conserved sequences diminishes MSL binding in vivo. MSL binding to these sites is roX RNA dependent, suggesting that complexes become competent for binding only after incorporation of roX RNAs. However, the roX RNA segments homologous to the DNA binding sites are not required, ruling out simple RNA-DNA complementarity as the primary targeting mechanism. Our results are consistent with a model in which nascent roX RNA assembly with MSL proteins is an early step in the initiation of dosage compensation.
In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of at least five proteins and two noncoding RNAs, roX1 and roX2. The roX RNAs function in targeting MSL complex to the X chromosome, and roX transgenes can nucleate spreading of the MSL complex into flanking chromatin when inserted on an autosome. An MSL-binding site (DHS, DNaseI hypersensitive site) has been identified in each roX gene. Here, we investigate the functions of the DHS using transgenic deletion analyses and reporter assays. We find that MSL interaction with the DHS counteracts constitutive repression at roX1, resulting in male-specific expression of roX1 RNA. Surprisingly, the DHS is not required for initiation of cis spreading of MSL complex, instead local transcription of roX RNAs correlates with extensive spreading. IntroductionTranscription in eukaryotes utilizes at least two levels of regulation: gene-specific control that operates locally on individual genes and global modulation of larger domains by chromatin composition or remodeling. Dosage compensation is an example of interplay between these two regulatory mechanisms that has evolved to make X-linked gene expression equivalent in males with one X chromosome and females with two. In Drosophila, dosage compensation is achieved by increasing the transcription of most X-linked genes two-fold in males (Lucchesi, 1998;Meller and Kuroda, 2002). This requires at least five proteins: MLE (maleless), MSL1, MSL2 and MSL3 (male-specific lethal 1, 2 and 3, respectively), and MOF (males absent on the first), and one of two roX (RNA on X) RNAs. MLE and MOF have enzymatic activities that are essential for dosage compensation: MLE is a DExH RNA helicase (Kuroda et al, 1991;Lee et al, 1997) and MOF is a MYST family histone acetyltransferase (Hilfiker et al, 1997;Akhtar and Becker, 2000;Smith et al, 2000). JIL-1, a histone H3 kinase, also associates with the MSL proteins (Jin et al, 2000). The MSL proteins, JIL-1 and roX RNAs bind in a precise pattern along the length of the male X chromosome, resulting in enrichment of chromatin modifications associated with hypertranscription, such as histone H4 acetylated at lysine 16 and H3 phosphorylated at serine 10 (Turner et al, 1992;Wang et al, 2001).The two noncoding RNAs, roX1 and roX2, are functionally redundant (Meller and Rattner, 2002) even though they have very little sequence homology and are distinct in size (3.7 kb for roX1 RNA versus 0.5-1.2 kb for roX2 RNA) (Amrein and Axel, 1997;Smith et al, 2000). Deletion of either roX gene has no effect on males. Missing both of them, however, results in male lethality (Meller et al, 1997;Meller and Rattner, 2002). The MSL-binding pattern on the X chromosome is drastically disrupted in these roX1roX2 double mutant males, suggesting that roX RNAs are important for correctly targeting MSL complex to the X chromosome. Both roX genes are located on X and overlap two of B35 chromatin entry sites (CESs), which are proposed to be high-affinity sites for MSL complexes du...
Dosage compensation in Drosophila is mediated by a histone-modifying complex that upregulates transcription of genes on the single male X chromosome. The male-specific lethal (MSL) complex contains at least five proteins and two noncoding roX (RNA on X) RNAs. The mechanism by which the MSL complex targets the X chromosome is not understood. Here we use a sensitized system to examine the function of roX genes on the X chromosome. In mutants that lack the NURF nucleosome remodeling complex, the male polytene X chromosome is severely distorted, appearing decondensed. This aberrant morphology is dependent on the MSL complex. Strikingly, roX mutations suppress the Nurf mutant phenotype regionally on the male X chromosome. Furthermore, a roX transgene induces disruption of local flanking autosomal chromatin in Nurf mutants. Taken together, these results demonstrate the potent capability of roX genes to organize large chromatin domains in cis, on the X chromosome. In addition to interacting functions at the level of chromosome morphology, we also find that NURF complex and MSL proteins have opposing effects on roX RNA transcription. Together, these results demonstrate the importance of a local balance between modifying activities that promote and antagonize chromatin compaction within defined chromatin domains in higher organisms.T HE compaction of eukaryotic DNA into nucleosomes and higher-order chromatin structure has profound effects on gene transcription. By modifying nucleosome structure or altering nucleosome positioning, histone-modifying enzymes and ATP-dependent chromatin-remodeling factors modulate chromatin architecture and thereby regulate gene expression. A major question is how the disparate activities of these chromatinorganizing enzymes are deployed and integrated.
The promoter-proximal pausing of RNA polymerase II (Pol II) plays a critical role in regulating metazoan gene transcription. Despite the prevalence of Pol II pausing across the metazoan genomes, little is known about the in vivo effect of Pol II pausing on vertebrate development. We use the emergence of hematopoietic stem cells (HSCs) in zebrafish embryos as a model to investigate the role of Pol II pausing in vertebrate organogenesis. Disrupting Pol II pausing machinery causes a severe reduction of HSC specification, a defect that can be effectively rescued by inhibiting Pol II elongation. In pausing-deficient embryos, the transforming growth factor β (TGFβ) signaling is elevated due to enhanced transcription elongation of key pathway genes, leading to HSC inhibition; in contrast, the interferon-γ (IFN-γ) signaling and its downstream effector Jak2/Stat3, which are required for HSC formation, are markedly attenuated owing to reduced chromatin accessibility on IFN-γ receptor genes. These findings reveal a novel transcription mechanism instructing HSC fate by pausing-mediated differential regulation of key signaling pathways.
Transcriptional regulators play critical roles in the regulation of cell fate during hematopoiesis. Previous studies in zebrafish have identified an essential role for the transcriptional intermediary factor TIF1γ in erythropoiesis through regulating the transcription elongation of erythroid genes. To study if TIF1γ plays a similar role in murine erythropoiesis and to assess its function in other blood lineages, we generated mouse models with hematopoietic deletion of TIF1γ. Our results showed a block in erythroid maturation in the bone marrow following tif1γ deletion that was compensated with enhanced spleen erythropoiesis. Further analyses revealed a defect in transcription elongation of erythroid genes in the bone marrow. In addition, loss of TIF1γ resulted in defects in other blood compartments, including a profound loss of B cells, a dramatic expansion of granulocytes and decreased HSC function. TIF1γ exerts its functions in a cell-autonomous manner as revealed by competitive transplantation experiments. Our study therefore demonstrates that TIF1γ plays essential roles in multiple murine blood lineages and that its function in transcription elongation is evolutionally conserved.
The pluripotency of embryonic stem cells (ESCs) relies on appropriate responsiveness to developmental cues. Promoter-proximal pausing of RNA polymerase II (Pol II) has been suggested to play a role in keeping genes poised for future activation. To identify the role of Pol II pausing in regulating ESC pluripotency, we have generated mouse ESCs carrying a mutation in the pause-inducing factor SPT5. Genomic studies reveal genome-wide reduction of paused Pol II caused by mutant SPT5 and further identify a tight correlation between pausing-mediated transcription effect and local chromatin environment. Functionally, this pausing-deficient SPT5 disrupts ESC differentiation upon removal of self-renewal signals. Thus, our study uncovers an important role of Pol II pausing in regulating ESC differentiation and suggests a model that Pol II pausing coordinates with epigenetic modification to influence transcription during mESC differentiation.
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