Summary paragraphThe mammalian genome is shaped by the expansion of repetitive elements that prov... more Summary paragraphThe mammalian genome is shaped by the expansion of repetitive elements that provide new regulatory networks for coordinated control of gene expression1 and genome folding2–4. Alu elements (AEs) are selectively retained close to the transcription start site of genes5, show protoenhancer functions6, correlate with the level of chromatin interactions7 and are recognized by transcription factor III (TFIIIC)8, but the relevance of all this is not clear. Here we report regulatory mechanisms that unveil a central role of AEs and TFIIIC in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of pre-marked AEs near cell cycle genes recruit TFIIIC to alter their chromatin accessibility via TFIIIC-mediated acetylation of histone H3 Lysine-18 (H3K18). This facilitates AEs contact with distant CTCF sites near other cell cycle genes promoters, which also become hyperacetylated at H3K18. These changes e...
In the last two decades, we have witnessed an impressive crescendo of non-coding RNA studies, due... more In the last two decades, we have witnessed an impressive crescendo of non-coding RNA studies, due to both the development of high-throughput RNA-sequencing strategies and an ever-increasing awareness of the involvement of newly discovered ncRNA classes in complex regulatory networks. Together with excitement for the possibility to explore previously unknown layers of gene regulation, these advancements led to the realization of the need for shared criteria of data collection and analysis and for novel integrative perspectives and tools aimed at making biological sense of very large bodies of molecular information. In the last few years, efforts to respond to this need have been devoted mainly to the regulatory interactions involving ncRNAs as direct or indirect regulators of protein-coding mRNAs. Such efforts resulted in the development of new computational tools, allowing the exploitation of the information spread in numerous different ncRNA data sets to interpret transcriptome cha...
Recent investigations on the non-protein-coding transcriptome of human cells have revealed previo... more Recent investigations on the non-protein-coding transcriptome of human cells have revealed previously hidden layers of gene regulation relying on regulatory non-protein-coding (nc) RNAs, including the widespread ncRNA-dependent regulation of epigenetic chromatin states and of mRNA translation and stability. However, despite its centrality, the epigenetic regulation of ncRNA genes has received relatively little attention. In this mini-review, we attempt to provide a synthetic account of recent literature suggesting an unexpected complexity in chromatin-dependent regulation of ncRNA gene transcription by the three human nuclear RNA polymerases. Emerging common features, like the heterogeneity of chromatin states within ncRNA multigene families and their influence on 3D genome organization, point to unexplored issues whose investigation could lead to a better understanding of the whole human epigenomic network.
Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to... more Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding—equilibrium and kinetics—by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin ...
In eukaryotes, RNA polymerase II (Pol II) is responsible for the synthesis of all mRNAs and myria... more In eukaryotes, RNA polymerase II (Pol II) is responsible for the synthesis of all mRNAs and myriads of short and long untranslated RNAs, whose fabrication involves close spatiotemporal coordination between transcription, RNA processing and chromatin modification. Crucial for such a coordination is an unusual C-terminal domain (CTD) of the Pol II largest subunit, made of tandem repetitions (26 in yeast, 52 in chordates) of the heptapeptide with the consensus sequence YSPTSPS. Although largely unstructured and with poor sequence content, the Pol II CTD derives its extraordinary functional versatility from the fact that each amino acid in the heptapeptide can be posttranslationally modified, and that different combinations of CTD covalent marks are specifically recognized by different protein binding partners. These features have led to propose the existence of a Pol II CTD code, but this expression is generally used by authors with some caution, revealed by the frequent use of quote marks for the word 'code'. Based on the theoretical framework of code biology, it is argued here that the Pol II CTD modification system meets the requirements of a true organic code, where different CTD modification states represent organic signs whose organic meanings are biological reactions contributing to the many facets of RNA biogenesis in coordination with RNA synthesis by Pol II. Importantly, the Pol II CTD code is instantiated by adaptor proteins possessing at least two distinct domains, one of which devoted to specific recognition of CTD modification profiles. Furthermore, code rules can be altered by experimental interchange of CTD recognition domains of different adaptor proteins, a fact arguing in favor of the arbitrariness, and thus bona fide character, of the Pol II CTD code. Since the growing family of CTD adaptors includes RNA binding proteins and histone modification complexes, the Pol II CTD code is by its nature integrated with other organic codes, in particular the splicing code and the histone code. These issues will be discussed taking into account fascinating developments in Pol II CTD research, like the discovery of novel modifications at non-consensus sites, the recently recognized CTD physicochemical properties favoring liquid-liquid phase separation, and the discovery that the Pol II CTD, originated before the divergence of most extant eukaryotic taxa, has expanded and diversified with developmental complexity in animals and plants.
Alternative splicing is a central component of human brain complexity; nonetheless, its regulator... more Alternative splicing is a central component of human brain complexity; nonetheless, its regulatory mechanisms are still largely unclear. In this work, we describe a novel non-coding (nc) RNA (named 17A) RNA polymerase (pol) III-dependent embedded in the human G-protein-coupled receptor 51 gene (GPR51, GABA B2 receptor). The stable expression of 17A in SHSY5Y neuroblastoma cells induces the synthesis of an alternative splicing isoform that abolish GABA B2 intracellular signaling (i.e., inhibition of cAMP accumulation and activation of K + channels). Indeed, 17A is expressed in human brain, and we report that it is upregulated in cerebral tissues derived from Alzheimer disease patients. We demonstrate that 17A expression in neuroblastoma cells enhances the secretion of amyloid β peptide (Aβ) and the Aβ x-42/Αβ x-40 peptide ratio and that its synthesis is induced in response to inflammatory stimuli. These data correlate, for the first time, the activity of a novel pol III-dependent ncRNA to alternative splicing events and, possibly, to neurodegeneration induced by abnormal GABA B function. We anticipate that further analysis of pol IIIdependent regulation of alternative splicing will disclose novel regulatory pathways associated to brain physiology and/or pathology.
Alu retroelements, whose retrotransposition requires prior transcription by RNA polymerase III to... more Alu retroelements, whose retrotransposition requires prior transcription by RNA polymerase III to generate Alu RNAs, represent the most numerous non-coding RNA (ncRNA) gene family in the human genome. Alu transcription is generally kept to extremely low levels by tight epigenetic silencing, but it has been reported to increase under different types of cell perturbation, such as viral infection and cancer. Alu RNAs, being able to act as gene expression modulators, may be directly involved in the mechanisms determining cellular behavior in such perturbed states. To directly address the regulatory potential of Alu RNAs, we generated IMR90 fibroblasts and HeLa cell lines stably overexpressing two slightly different Alu RNAs, and analyzed genome-wide the expression changes of protein-coding genes through RNA-sequencing. Among the genes that were upregulated or downregulated in response to Alu overexpression in IMR90, but not in HeLa cells, we found a highly significant enrichment of path...
Biochemical and Biophysical Research Communications, 2006
The SNR52 gene, coding for a box C/D snoRNA, is the only snoRNA gene transcribed by RNA polymeras... more The SNR52 gene, coding for a box C/D snoRNA, is the only snoRNA gene transcribed by RNA polymerase (Pol) III in Saccharomyces cerevisiae. Pol III transcription generates a precisely terminated primary transcript that undergoes extensive 5 0-end processing. Here, we show that mutations of the box C/D core motif required for snoRNP assembly compromise 5 0-end maturation of the SNR52 snoRNA. Upstream processing was also impaired by specific depletion of either Nop1p or Nop58p snoRNP proteins. We further show that the nuclear exosome is required for 3 0-end maturation of SNR52 snoRNA, at variance with all the other known Pol III transcripts. Our data suggest a functional coupling between snoRNP assembly and 5 0-end maturation of independently transcribed box C/D snoRNAs.
Retrotransposons, a large and diverse class of transposable elements that are still active in hum... more Retrotransposons, a large and diverse class of transposable elements that are still active in humans, represent a remarkable force of genomic innovation underlying mammalian evolution. Among the features distinguishing mammals from all other vertebrates, the presence of a neocortex with a peculiar neuronal organization, composition and connectivity is perhaps the one that, by affecting the cognitive abilities of mammals, contributed mostly to their evolutionary success. Among mammals, hominids and especially humans display an extraordinarily expanded cortical volume, an enrichment of the repertoire of neural cell types and more elaborate patterns of neuronal connectivity. Retrotransposon-derived sequences have recently been implicated in multiple layers of gene regulation in the brain, from transcriptional and post-transcriptional control to both local and large-scale three-dimensional chromatin organization. Accordingly, an increasing variety of neurodevelopmental and neurodegenera...
International journal of environmental research and public health, Jan 12, 2018
Benzene, a known human carcinogen, and methyl -butyl ether (MTBE), not classifiable as to its car... more Benzene, a known human carcinogen, and methyl -butyl ether (MTBE), not classifiable as to its carcinogenicity, are fuel-related pollutants. This study investigated the effect of these chemicals on epigenetic and transcriptional alterations in DNA repetitive elements. In 89 petrol station workers and 90 non-occupationally exposed subjects the transcriptional activity of retrotransposons (LINE-1, Alu), the methylation on repeated-element DNA, and of H3K9 histone, were investigated in peripheral blood lymphocytes. Median work shift exposure to benzene and MTBE was 59 and 408 µg/m³ in petrol station workers, and 4 and 3.5 µg/m³, in controls. Urinary benzene (BEN-U), -phenylmercapturic acid, and MTBE were significantly higher in workers than in controls, while -muconic acid (-MA) was comparable between the two groups. Increased BEN-U was associated with increased and expression; moreover, increased -MA was associated with increased and and LINE-1 (L1)-5'UTR expression. Among repetiti...
With more than 500,000 copies, mammalian-wide interspersed repeats (MIRs), a subgroup of SINEs, r... more With more than 500,000 copies, mammalian-wide interspersed repeats (MIRs), a subgroup of SINEs, represent $2.5% of the human genome and one of the most numerous family of potential targets for the RNA polymerase (Pol) III transcription machinery. Since MIR elements ceased to amplify $130 myr ago, previous studies primarily focused on their genomic impact, while the issue of their expression has not been extensively addressed. We applied a dedicated bioinformatic pipeline to ENCODE RNA-Seq datasets of seven human cell lines and, for the first time, we were able to define the Pol III-driven MIR transcriptome at single-locus resolution. While the majority of Pol III-transcribed MIR elements are cell-specific, we discovered a small set of ubiquitously transcribed MIRs mapping within Pol II-transcribed genes in antisense orientation that could influence the expression of the overlapping gene. We also identified novel Pol III-transcribed ncRNAs, deriving from transcription of annotated MIR fragments flanked by unique MIR-unrelated sequences, and confirmed the role of Pol III-specific internal promoter elements in MIR transcription. Besides demonstrating widespread transcription at these retrotranspositionally inactive elements in human cells, the ability to profile MIR expression at single-locus resolution will facilitate their study in different cell types and states including pathological alterations.
In Saccharomyces cerevisiae, a group of more than 200 co-regulated genes (Ribi genes) is involved... more In Saccharomyces cerevisiae, a group of more than 200 co-regulated genes (Ribi genes) is involved in ribosome biogenesis. This regulon has recently been shown to rely on a small set of transcriptional regulators (mainly Abf1, but also Reb1, Tbf1 and Rap1) previously referred to as general regulatory factors (GRFs) because of their widespread binding and action at many promoters and other specialized genomic regions. Intriguingly, Abf1 binding to Ribi genes is differentially modulated in response to distinct nutrition signaling pathways. Such a dynamic promoter association has the potential to orchestrate both activation and repression of Ribi genes in synergy with neighboring regulatory sites and through the functional interplay of histone acetyltransferases and deacetylases.
In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for eit... more In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for either Rap1 or Abf1 transcription factors. Unlike Rap1associated promoters, the small cohort of Abf1dependent RPGs (Abf1-RPGs) has not been extensively investigated. We show that RPL3, RPL4B, RPP1A, RPS22B and RPS28A/B share a common promoter architecture, with an Abf1 site upstream of a conserved element matching the sequence recognized by Fhl1, a transcription factor which together with Ifh1 orchestrates Rap1-associated RPG regulation. Abf1 and Fhl1 promoter association was confirmed by ChIP and/or gel retardation assays. Mutational analysis revealed a more severe requirement of Abf1 than Fhl1 binding sites for RPG transcription. In the case of RPS22B an unusual Tbf1 binding site promoted both RPS22B and intron-hosted SNR44 expression. Abf1-RPG down-regulation upon TOR pathway inhibition was much attenuated at defective mutant promoters unable to bind Abf1. TORC1 inactivation caused the expected reduction of Ifh1 occupancy at RPS22B and RPL3 promoters, but unexpectedly it entailed largely increased Abf1 association with Abf1-RPG promoters. We present evidence that Abf1 recruitment upon nutritional stress, also observed for representative ribosome biogenesis genes, favours RPG transcriptional rescue upon nutrient replenishment, thus pointing to nutrientregulated Abf1 dynamics at promoters as a novel mechanism in ribosome biogenesis control.
In Saccharomyces cerevisiae, the large majority of the genes coding for cytoplasmic ribosomal pro... more In Saccharomyces cerevisiae, the large majority of the genes coding for cytoplasmic ribosomal proteins (RPs) depend on the general regulatory factor Rap1 for their transcription, but a small cohort of them relies on Abf1 regulatory activity. A recent study showed that unlike Rap1, whose association with RP gene promoters is not affected by environmental changes causing RP gene repression/reactivation, Abf1 association with both RP gene and ribosome biogenesis (Ribi) gene promoters dynamically responds to changes in growth conditions. This observation changes the paradigm of general regulatory factors as relatively static DNA-binding proteins constitutively bound to highly active promoters, and point to Abf1, which binds hundreds of non-RPG promoters within the yeast genome, as a possible key regulatory switch in nutrient- and stress-dependent transcriptional modulation. Moreover, the frequent presence of Abf1 binding sites in the promoters of mitochondrial RP genes evokes the possib...
Ribosome biogenesis in Saccharomyces cerevisiae involves a regulon of >200 genes (Ribi genes) coo... more Ribosome biogenesis in Saccharomyces cerevisiae involves a regulon of >200 genes (Ribi genes) coordinately regulated in response to nutrient availability and cellular growth rate. Two cis-acting elements called PAC and RRPE are known to mediate Ribi gene repression in response to nutritional downshift. Here, we show that most Ribi gene promoters also contain binding sites for one or more General Regulatory Factors (GRFs), most frequently Abf1 and Reb1, and that these factors are enriched in vivo at Ribi promoters. Abf1/Reb1/Tbf1 promoter association was required for full Ribi gene expression in rich medium and for its modulation in response to glucose starvation, characterized by a rapid drop followed by slow recovery. Such a response did not entail changes in Abf1 occupancy, but it was paralleled by a quick increase, followed by slow decrease, in Rpd3L histone deacetylase occupancy. Remarkably, Abf1 site disruption also abolished Rpd3L complex recruitment in response to starvation. Extensive mutational analysis of the DBP7 promoter revealed a complex interplay of Tbf1 sites, PAC and RRPE in the transcriptional regulation of this Ribi gene. Our observations point to GRFs as new multifaceted players in Ribi gene regulation both during exponential growth and under repressive conditions.
Biochemical and Biophysical Research Communications, 2016
Hydroquinone (HQ) is an important benzene-derived metabolite associated with acute myelogenous le... more Hydroquinone (HQ) is an important benzene-derived metabolite associated with acute myelogenous leukemia risk. Although altered DNA methylation has been reported in both benzene-exposed human subjects and HQ-exposed cultured cells, the inventory of benzene metabolite effects on the epigenome is only starting to be established. In this study, we used a monocytic leukemia cell line (THP-1) and hematopoietic stem cells (HSCs) from cord blood to investigate the effects of HQ treatment on the expression of the three most important families of retrotransposons in the human genome: LINE-1, Alu and Endogenous retroviruses (HERVs), that are normally subjected to tight epigenetic silencing. We found a clear tendency towards increased retrotransposon expression in response to HQ exposure, more pronounced in the case of LINE-1 and HERV. Such a partial loss of silencing, however, was generally not associated with HQ-induced DNA hypomethylation. On the other hand, retroelement derepression was also observed in the same cells in response to the hypomethylating agent decitabine. These observations suggest the existence of different types of epigenetic switches operating at human retroelements, and point to retroelement activation in response to benzene-derived metabolites as a novel factor deserving attention in benzenecarcinogenesis studies.
Summary paragraphThe mammalian genome is shaped by the expansion of repetitive elements that prov... more Summary paragraphThe mammalian genome is shaped by the expansion of repetitive elements that provide new regulatory networks for coordinated control of gene expression1 and genome folding2–4. Alu elements (AEs) are selectively retained close to the transcription start site of genes5, show protoenhancer functions6, correlate with the level of chromatin interactions7 and are recognized by transcription factor III (TFIIIC)8, but the relevance of all this is not clear. Here we report regulatory mechanisms that unveil a central role of AEs and TFIIIC in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of pre-marked AEs near cell cycle genes recruit TFIIIC to alter their chromatin accessibility via TFIIIC-mediated acetylation of histone H3 Lysine-18 (H3K18). This facilitates AEs contact with distant CTCF sites near other cell cycle genes promoters, which also become hyperacetylated at H3K18. These changes e...
In the last two decades, we have witnessed an impressive crescendo of non-coding RNA studies, due... more In the last two decades, we have witnessed an impressive crescendo of non-coding RNA studies, due to both the development of high-throughput RNA-sequencing strategies and an ever-increasing awareness of the involvement of newly discovered ncRNA classes in complex regulatory networks. Together with excitement for the possibility to explore previously unknown layers of gene regulation, these advancements led to the realization of the need for shared criteria of data collection and analysis and for novel integrative perspectives and tools aimed at making biological sense of very large bodies of molecular information. In the last few years, efforts to respond to this need have been devoted mainly to the regulatory interactions involving ncRNAs as direct or indirect regulators of protein-coding mRNAs. Such efforts resulted in the development of new computational tools, allowing the exploitation of the information spread in numerous different ncRNA data sets to interpret transcriptome cha...
Recent investigations on the non-protein-coding transcriptome of human cells have revealed previo... more Recent investigations on the non-protein-coding transcriptome of human cells have revealed previously hidden layers of gene regulation relying on regulatory non-protein-coding (nc) RNAs, including the widespread ncRNA-dependent regulation of epigenetic chromatin states and of mRNA translation and stability. However, despite its centrality, the epigenetic regulation of ncRNA genes has received relatively little attention. In this mini-review, we attempt to provide a synthetic account of recent literature suggesting an unexpected complexity in chromatin-dependent regulation of ncRNA gene transcription by the three human nuclear RNA polymerases. Emerging common features, like the heterogeneity of chromatin states within ncRNA multigene families and their influence on 3D genome organization, point to unexplored issues whose investigation could lead to a better understanding of the whole human epigenomic network.
Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to... more Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding—equilibrium and kinetics—by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin ...
In eukaryotes, RNA polymerase II (Pol II) is responsible for the synthesis of all mRNAs and myria... more In eukaryotes, RNA polymerase II (Pol II) is responsible for the synthesis of all mRNAs and myriads of short and long untranslated RNAs, whose fabrication involves close spatiotemporal coordination between transcription, RNA processing and chromatin modification. Crucial for such a coordination is an unusual C-terminal domain (CTD) of the Pol II largest subunit, made of tandem repetitions (26 in yeast, 52 in chordates) of the heptapeptide with the consensus sequence YSPTSPS. Although largely unstructured and with poor sequence content, the Pol II CTD derives its extraordinary functional versatility from the fact that each amino acid in the heptapeptide can be posttranslationally modified, and that different combinations of CTD covalent marks are specifically recognized by different protein binding partners. These features have led to propose the existence of a Pol II CTD code, but this expression is generally used by authors with some caution, revealed by the frequent use of quote marks for the word 'code'. Based on the theoretical framework of code biology, it is argued here that the Pol II CTD modification system meets the requirements of a true organic code, where different CTD modification states represent organic signs whose organic meanings are biological reactions contributing to the many facets of RNA biogenesis in coordination with RNA synthesis by Pol II. Importantly, the Pol II CTD code is instantiated by adaptor proteins possessing at least two distinct domains, one of which devoted to specific recognition of CTD modification profiles. Furthermore, code rules can be altered by experimental interchange of CTD recognition domains of different adaptor proteins, a fact arguing in favor of the arbitrariness, and thus bona fide character, of the Pol II CTD code. Since the growing family of CTD adaptors includes RNA binding proteins and histone modification complexes, the Pol II CTD code is by its nature integrated with other organic codes, in particular the splicing code and the histone code. These issues will be discussed taking into account fascinating developments in Pol II CTD research, like the discovery of novel modifications at non-consensus sites, the recently recognized CTD physicochemical properties favoring liquid-liquid phase separation, and the discovery that the Pol II CTD, originated before the divergence of most extant eukaryotic taxa, has expanded and diversified with developmental complexity in animals and plants.
Alternative splicing is a central component of human brain complexity; nonetheless, its regulator... more Alternative splicing is a central component of human brain complexity; nonetheless, its regulatory mechanisms are still largely unclear. In this work, we describe a novel non-coding (nc) RNA (named 17A) RNA polymerase (pol) III-dependent embedded in the human G-protein-coupled receptor 51 gene (GPR51, GABA B2 receptor). The stable expression of 17A in SHSY5Y neuroblastoma cells induces the synthesis of an alternative splicing isoform that abolish GABA B2 intracellular signaling (i.e., inhibition of cAMP accumulation and activation of K + channels). Indeed, 17A is expressed in human brain, and we report that it is upregulated in cerebral tissues derived from Alzheimer disease patients. We demonstrate that 17A expression in neuroblastoma cells enhances the secretion of amyloid β peptide (Aβ) and the Aβ x-42/Αβ x-40 peptide ratio and that its synthesis is induced in response to inflammatory stimuli. These data correlate, for the first time, the activity of a novel pol III-dependent ncRNA to alternative splicing events and, possibly, to neurodegeneration induced by abnormal GABA B function. We anticipate that further analysis of pol IIIdependent regulation of alternative splicing will disclose novel regulatory pathways associated to brain physiology and/or pathology.
Alu retroelements, whose retrotransposition requires prior transcription by RNA polymerase III to... more Alu retroelements, whose retrotransposition requires prior transcription by RNA polymerase III to generate Alu RNAs, represent the most numerous non-coding RNA (ncRNA) gene family in the human genome. Alu transcription is generally kept to extremely low levels by tight epigenetic silencing, but it has been reported to increase under different types of cell perturbation, such as viral infection and cancer. Alu RNAs, being able to act as gene expression modulators, may be directly involved in the mechanisms determining cellular behavior in such perturbed states. To directly address the regulatory potential of Alu RNAs, we generated IMR90 fibroblasts and HeLa cell lines stably overexpressing two slightly different Alu RNAs, and analyzed genome-wide the expression changes of protein-coding genes through RNA-sequencing. Among the genes that were upregulated or downregulated in response to Alu overexpression in IMR90, but not in HeLa cells, we found a highly significant enrichment of path...
Biochemical and Biophysical Research Communications, 2006
The SNR52 gene, coding for a box C/D snoRNA, is the only snoRNA gene transcribed by RNA polymeras... more The SNR52 gene, coding for a box C/D snoRNA, is the only snoRNA gene transcribed by RNA polymerase (Pol) III in Saccharomyces cerevisiae. Pol III transcription generates a precisely terminated primary transcript that undergoes extensive 5 0-end processing. Here, we show that mutations of the box C/D core motif required for snoRNP assembly compromise 5 0-end maturation of the SNR52 snoRNA. Upstream processing was also impaired by specific depletion of either Nop1p or Nop58p snoRNP proteins. We further show that the nuclear exosome is required for 3 0-end maturation of SNR52 snoRNA, at variance with all the other known Pol III transcripts. Our data suggest a functional coupling between snoRNP assembly and 5 0-end maturation of independently transcribed box C/D snoRNAs.
Retrotransposons, a large and diverse class of transposable elements that are still active in hum... more Retrotransposons, a large and diverse class of transposable elements that are still active in humans, represent a remarkable force of genomic innovation underlying mammalian evolution. Among the features distinguishing mammals from all other vertebrates, the presence of a neocortex with a peculiar neuronal organization, composition and connectivity is perhaps the one that, by affecting the cognitive abilities of mammals, contributed mostly to their evolutionary success. Among mammals, hominids and especially humans display an extraordinarily expanded cortical volume, an enrichment of the repertoire of neural cell types and more elaborate patterns of neuronal connectivity. Retrotransposon-derived sequences have recently been implicated in multiple layers of gene regulation in the brain, from transcriptional and post-transcriptional control to both local and large-scale three-dimensional chromatin organization. Accordingly, an increasing variety of neurodevelopmental and neurodegenera...
International journal of environmental research and public health, Jan 12, 2018
Benzene, a known human carcinogen, and methyl -butyl ether (MTBE), not classifiable as to its car... more Benzene, a known human carcinogen, and methyl -butyl ether (MTBE), not classifiable as to its carcinogenicity, are fuel-related pollutants. This study investigated the effect of these chemicals on epigenetic and transcriptional alterations in DNA repetitive elements. In 89 petrol station workers and 90 non-occupationally exposed subjects the transcriptional activity of retrotransposons (LINE-1, Alu), the methylation on repeated-element DNA, and of H3K9 histone, were investigated in peripheral blood lymphocytes. Median work shift exposure to benzene and MTBE was 59 and 408 µg/m³ in petrol station workers, and 4 and 3.5 µg/m³, in controls. Urinary benzene (BEN-U), -phenylmercapturic acid, and MTBE were significantly higher in workers than in controls, while -muconic acid (-MA) was comparable between the two groups. Increased BEN-U was associated with increased and expression; moreover, increased -MA was associated with increased and and LINE-1 (L1)-5'UTR expression. Among repetiti...
With more than 500,000 copies, mammalian-wide interspersed repeats (MIRs), a subgroup of SINEs, r... more With more than 500,000 copies, mammalian-wide interspersed repeats (MIRs), a subgroup of SINEs, represent $2.5% of the human genome and one of the most numerous family of potential targets for the RNA polymerase (Pol) III transcription machinery. Since MIR elements ceased to amplify $130 myr ago, previous studies primarily focused on their genomic impact, while the issue of their expression has not been extensively addressed. We applied a dedicated bioinformatic pipeline to ENCODE RNA-Seq datasets of seven human cell lines and, for the first time, we were able to define the Pol III-driven MIR transcriptome at single-locus resolution. While the majority of Pol III-transcribed MIR elements are cell-specific, we discovered a small set of ubiquitously transcribed MIRs mapping within Pol II-transcribed genes in antisense orientation that could influence the expression of the overlapping gene. We also identified novel Pol III-transcribed ncRNAs, deriving from transcription of annotated MIR fragments flanked by unique MIR-unrelated sequences, and confirmed the role of Pol III-specific internal promoter elements in MIR transcription. Besides demonstrating widespread transcription at these retrotranspositionally inactive elements in human cells, the ability to profile MIR expression at single-locus resolution will facilitate their study in different cell types and states including pathological alterations.
In Saccharomyces cerevisiae, a group of more than 200 co-regulated genes (Ribi genes) is involved... more In Saccharomyces cerevisiae, a group of more than 200 co-regulated genes (Ribi genes) is involved in ribosome biogenesis. This regulon has recently been shown to rely on a small set of transcriptional regulators (mainly Abf1, but also Reb1, Tbf1 and Rap1) previously referred to as general regulatory factors (GRFs) because of their widespread binding and action at many promoters and other specialized genomic regions. Intriguingly, Abf1 binding to Ribi genes is differentially modulated in response to distinct nutrition signaling pathways. Such a dynamic promoter association has the potential to orchestrate both activation and repression of Ribi genes in synergy with neighboring regulatory sites and through the functional interplay of histone acetyltransferases and deacetylases.
In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for eit... more In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for either Rap1 or Abf1 transcription factors. Unlike Rap1associated promoters, the small cohort of Abf1dependent RPGs (Abf1-RPGs) has not been extensively investigated. We show that RPL3, RPL4B, RPP1A, RPS22B and RPS28A/B share a common promoter architecture, with an Abf1 site upstream of a conserved element matching the sequence recognized by Fhl1, a transcription factor which together with Ifh1 orchestrates Rap1-associated RPG regulation. Abf1 and Fhl1 promoter association was confirmed by ChIP and/or gel retardation assays. Mutational analysis revealed a more severe requirement of Abf1 than Fhl1 binding sites for RPG transcription. In the case of RPS22B an unusual Tbf1 binding site promoted both RPS22B and intron-hosted SNR44 expression. Abf1-RPG down-regulation upon TOR pathway inhibition was much attenuated at defective mutant promoters unable to bind Abf1. TORC1 inactivation caused the expected reduction of Ifh1 occupancy at RPS22B and RPL3 promoters, but unexpectedly it entailed largely increased Abf1 association with Abf1-RPG promoters. We present evidence that Abf1 recruitment upon nutritional stress, also observed for representative ribosome biogenesis genes, favours RPG transcriptional rescue upon nutrient replenishment, thus pointing to nutrientregulated Abf1 dynamics at promoters as a novel mechanism in ribosome biogenesis control.
In Saccharomyces cerevisiae, the large majority of the genes coding for cytoplasmic ribosomal pro... more In Saccharomyces cerevisiae, the large majority of the genes coding for cytoplasmic ribosomal proteins (RPs) depend on the general regulatory factor Rap1 for their transcription, but a small cohort of them relies on Abf1 regulatory activity. A recent study showed that unlike Rap1, whose association with RP gene promoters is not affected by environmental changes causing RP gene repression/reactivation, Abf1 association with both RP gene and ribosome biogenesis (Ribi) gene promoters dynamically responds to changes in growth conditions. This observation changes the paradigm of general regulatory factors as relatively static DNA-binding proteins constitutively bound to highly active promoters, and point to Abf1, which binds hundreds of non-RPG promoters within the yeast genome, as a possible key regulatory switch in nutrient- and stress-dependent transcriptional modulation. Moreover, the frequent presence of Abf1 binding sites in the promoters of mitochondrial RP genes evokes the possib...
Ribosome biogenesis in Saccharomyces cerevisiae involves a regulon of >200 genes (Ribi genes) coo... more Ribosome biogenesis in Saccharomyces cerevisiae involves a regulon of >200 genes (Ribi genes) coordinately regulated in response to nutrient availability and cellular growth rate. Two cis-acting elements called PAC and RRPE are known to mediate Ribi gene repression in response to nutritional downshift. Here, we show that most Ribi gene promoters also contain binding sites for one or more General Regulatory Factors (GRFs), most frequently Abf1 and Reb1, and that these factors are enriched in vivo at Ribi promoters. Abf1/Reb1/Tbf1 promoter association was required for full Ribi gene expression in rich medium and for its modulation in response to glucose starvation, characterized by a rapid drop followed by slow recovery. Such a response did not entail changes in Abf1 occupancy, but it was paralleled by a quick increase, followed by slow decrease, in Rpd3L histone deacetylase occupancy. Remarkably, Abf1 site disruption also abolished Rpd3L complex recruitment in response to starvation. Extensive mutational analysis of the DBP7 promoter revealed a complex interplay of Tbf1 sites, PAC and RRPE in the transcriptional regulation of this Ribi gene. Our observations point to GRFs as new multifaceted players in Ribi gene regulation both during exponential growth and under repressive conditions.
Biochemical and Biophysical Research Communications, 2016
Hydroquinone (HQ) is an important benzene-derived metabolite associated with acute myelogenous le... more Hydroquinone (HQ) is an important benzene-derived metabolite associated with acute myelogenous leukemia risk. Although altered DNA methylation has been reported in both benzene-exposed human subjects and HQ-exposed cultured cells, the inventory of benzene metabolite effects on the epigenome is only starting to be established. In this study, we used a monocytic leukemia cell line (THP-1) and hematopoietic stem cells (HSCs) from cord blood to investigate the effects of HQ treatment on the expression of the three most important families of retrotransposons in the human genome: LINE-1, Alu and Endogenous retroviruses (HERVs), that are normally subjected to tight epigenetic silencing. We found a clear tendency towards increased retrotransposon expression in response to HQ exposure, more pronounced in the case of LINE-1 and HERV. Such a partial loss of silencing, however, was generally not associated with HQ-induced DNA hypomethylation. On the other hand, retroelement derepression was also observed in the same cells in response to the hypomethylating agent decitabine. These observations suggest the existence of different types of epigenetic switches operating at human retroelements, and point to retroelement activation in response to benzene-derived metabolites as a novel factor deserving attention in benzenecarcinogenesis studies.
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Papers by Giorgio Dieci