Publications by authors named "Angad Garg"

12 Publications

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Structure-function analysis of fission yeast cleavage and polyadenylation factor (CPF) subunit Ppn1 and its interactions with Dis2 and Swd22.

PLoS Genet 2021 Mar 12;17(3):e1009452. Epub 2021 Mar 12.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York, United States of America.

Fission yeast Cleavage and Polyadenylation Factor (CPF), a 13-subunit complex, executes the cotranscriptional 3' processing of RNA polymerase II (Pol2) transcripts that precedes transcription termination. The three-subunit DPS sub-complex of CPF, consisting of a PP1-type phosphoprotein phosphatase Dis2, a WD-repeat protein Swd22, and a putative phosphatase regulatory factor Ppn1, associates with the CPF core to form the holo-CPF assembly. Here we probed the functional, physical, and genetic interactions of DPS by focusing on the Ppn1 subunit, which mediates association of DPS with the core. Transcriptional profiling by RNA-seq defined limited but highly concordant sets of protein-coding genes that were dysregulated in ppn1Δ, swd22Δ and dis2Δ cells, which included the DPSΔ down-regulated phosphate homeostasis genes pho1 and pho84 that are controlled by lncRNA-mediated transcriptional interference. Essential and inessential modules of the 710-aa Ppn1 protein were defined by testing the effects of Ppn1 truncations in multiple genetic backgrounds in which Ppn1 is required for growth. An N-terminal 172-aa disordered region was dispensable and its deletion alleviated hypomorphic phenotypes caused by deleting C-terminal aa 640-710. A TFIIS-like domain (aa 173-330) was not required for viability but was important for Ppn1 activity in phosphate homeostasis. Distinct sites within Ppn1 for binding to Dis2 (spanning Ppn1 aa 506 to 532) and Swd22 (from Ppn1 aa 533 to 578) were demarcated by yeast two-hybrid assays. Dis2 interaction-defective missense mutants of full-length Ppn1 (that retained Swd22 interaction) were employed to show that binding to Dis2 (or its paralog Sds21) was necessary for Ppn1 biological activity. Ppn1 function was severely compromised by missense mutations that selectively affected its binding to Swd22.
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http://dx.doi.org/10.1371/journal.pgen.1009452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990198PMC
March 2021

Transcriptional profiling of fission yeast RNA polymerase II CTD mutants.

RNA 2021 Feb 12. Epub 2021 Feb 12.

Sloan Kettering Institute;

The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol2) consists of tandem repeats of a consensus heptapeptide Y S P T S P S The CTD recruits numerous proteins that drive or regulate gene expression. The trafficking of CTD-interacting proteins is orchestrated by remodeling CTD primary structure via Ser/Thr/Tyr phosphorylation and proline isomerization, which collectively inscribe a CTD code. The fission yeast CTD consists of 29 heptad repeats. To decipher the output of the fission yeast CTD code, we genetically manipulated CTD length and amino acid content and then gauged the effects of these changes on gene expression. Whereas deleting 11 consensus heptads has no obvious effect on fission yeast growth, RNA-seq revealed that 25% of the protein-coding transcripts were dysregulated by CTD truncation. We profiled the transcriptomes of full-length CTD mutants, in which: all Tyr1 residues were replaced by Phe; all Ser2, Thr4, or Ser7 positions were changed to Ala; and half of the essential CTD code "letters" Pro3, Ser5, and Pro6 were mutated to Ala. Overlapping RNA-seq profiles suggested that a quarter of the complement of up-regulated mRNAs and half of the down-regulated mRNAs seen in full-length CTD mutants might be attributable to a decrement in wild-type CTD heptad number. Concordant mutant-specific transcriptional profiles were observed for , , and cells, and for and cells, suggesting that Tyr1-Ser2-Thr4 and Ser5-Pro6 comprise distinct "words" in the fission yeast CTD code. The phosphate regulon, which is repressed by lncRNA-mediated transcription interference, is de-repressed by CTD mutations P6•P6A and S5•S5A. De-repression of pho1 in P6•P6A and S5•S5A cells depends on cleavage and polyadenylation factor subunits Swd22 and Ppn1 and transcription termination factor Rhn1, signifying that Pro6 and Ser5 mutations elicit precocious lncRNA 3'-processing/termination.
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http://dx.doi.org/10.1261/rna.078682.121DOI Listing
February 2021

A genetic screen for suppressors of hyper-repression of the fission yeast PHO regulon by Pol2 CTD mutation T4A implicates inositol 1-pyrophosphates as agonists of precocious lncRNA transcription termination.

Nucleic Acids Res 2020 11;48(19):10739-10752

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA.

Fission yeast phosphate homeostasis genes are repressed in phosphate-rich medium by transcription of upstream lncRNAs that interferes with activation of the flanking mRNA promoters. lncRNA control of PHO gene expression is influenced by the Thr4 phospho-site in the RNA polymerase II CTD and the 3' processing/termination factors CPF and Rhn1, mutations of which result in hyper-repression of the PHO regulon. Here, we performed a forward genetic screen for mutations that de-repress Pho1 acid phosphatase expression in CTD-T4A cells. Sequencing of 18 independent STF (Suppressor of Threonine Four) isolates revealed, in every case, a mutation in the C-terminal pyrophosphatase domain of Asp1, a bifunctional inositol pyrophosphate (IPP) kinase/pyrophosphatase that interconverts 5-IP7 and 1,5-IP8. Focused characterization of two STF strains identified 51 coding genes coordinately upregulated vis-à-vis the parental T4A strain, including all three PHO regulon genes (pho1, pho84, tgp1). Whereas these STF alleles-asp1-386(Stop) and asp1-493(Stop)-were lethal in a wild-type CTD background, they were viable in combination with mutations in CPF and Rhn1, in which context Pho1 was also de-repressed. Our findings implicate Asp1 pyrophosphatase in constraining 1,5-IP8 or 1-IP7 synthesis by Asp1 kinase, without which 1-IPPs can accumulate to toxic levels that elicit precocious termination by CPF/Rhn1.
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http://dx.doi.org/10.1093/nar/gkaa776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641756PMC
November 2020

A lncRNA-regulated gene expression system with rapid induction kinetics in the fission yeast .

Authors:
Angad Garg

RNA 2020 11 11;26(11):1743-1752. Epub 2020 Aug 11.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA

The fission yeast is an excellent model organism for the study of eukaryotic cellular physiology. The organism is genetically tractable and several tools to study the functions of individual genes are available. One such tool is regulatable gene expression and overproduction of proteins. Limitations of currently available overexpression systems include delay in expression after induction, narrow dynamic range, and system-wide changes due to induction conditions. Here I describe a new long noncoding RNA (lncRNA)-regulated, thiamine-inducible expression system that integrates lncRNA-based transcriptional interference at the fission yeast promoter with the fast repression kinetics of the thiamine-repressible promoter. This hybrid system has rapid induction kinetics, broad dynamic range, and tunable expression via thiamine concentration. The lncRNA-regulated thiamine-inducible system will be advantageous for the study of individual genes and for potential applications in the production of heterologous proteins in fission yeast.
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http://dx.doi.org/10.1261/rna.076000.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566572PMC
November 2020

Genetic interactions and transcriptomics implicate fission yeast CTD prolyl isomerase Pin1 as an agent of RNA 3' processing and transcription termination that functions via its effects on CTD phosphatase Ssu72.

Nucleic Acids Res 2020 05;48(9):4811-4826

Dept. of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA.

The phosphorylation pattern of Pol2 CTD Y1S2P3T4S5P6S7 repeats comprises an informational code coordinating transcription and RNA processing. cis-trans isomerization of CTD prolines expands the scope of the code in ways that are not well understood. Here we address this issue via analysis of fission yeast peptidyl-prolyl isomerase Pin1. A pin1Δ allele that does not affect growth per se is lethal in the absence of cleavage-polyadenylation factor (CPF) subunits Ppn1 and Swd22 and elicits growth defects absent CPF subunits Ctf1 and Dis2 and termination factor Rhn1. Whereas CTD S2A, T4A, and S7A mutants thrive in combination with pin1Δ, a Y1F mutant does not, nor do CTD mutants in which half the Pro3 or Pro6 residues are replaced by alanine. Phosphate-acquisition genes pho1, pho84 and tgp1 are repressed by upstream lncRNAs and are sensitive to changes in lncRNA 3' processing/termination. pin1Δ hyper-represses PHO gene expression and erases the de-repressive effect of CTD-S7A. Transcriptional profiling delineated sets of 56 and 22 protein-coding genes that are down-regulated and up-regulated in pin1Δ cells, respectively, 77% and 100% of which are downregulated/upregulated when the cis-proline-dependent Ssu72 CTD phosphatase is inactivated. Our results implicate Pin1 as a positive effector of 3' processing/termination that acts via Ssu72.
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http://dx.doi.org/10.1093/nar/gkaa212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229847PMC
May 2020

Inositol pyrophosphates impact phosphate homeostasis via modulation of RNA 3' processing and transcription termination.

Nucleic Acids Res 2019 09;47(16):8452-8469

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA.

Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here, we show that phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3'-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of the Asp1 and Aps1 IPP pyrophosphatases is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1Δ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3'-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.
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http://dx.doi.org/10.1093/nar/gkz567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895273PMC
September 2019

Structure of Fission Yeast Transcription Factor Pho7 Bound to Promoter DNA and Effect of Pho7 Mutations on DNA Binding and Phosphate Homeostasis.

Mol Cell Biol 2019 07 13;39(13). Epub 2019 Jun 13.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA

Pho7 is the fission yeast ZnCys transcriptional factor that drives a response to phosphate starvation in which phosphate acquisition genes are upregulated. Here we report a crystal structure at 1.6-Å resolution of the Pho7 DNA-binding domain (DBD) bound at its target site 2 in the promoter (5'-TCGGAAATTAAAAA). Comparison to the previously reported structure of Pho7 DBD in complex with its binding site in the promoter (5'-TCGGACATTCAAAT) reveals shared determinants of target site specificity as well as variations in the protein-DNA interface that accommodate different promoter DNA sequences. Mutagenesis of Pho7 amino acids at the DNA interface identified nucleobase contacts at the periphery of the footprint that are essential for the induction of expression in response to phosphate starvation and for Pho7 binding to site 1 in the promoter.
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http://dx.doi.org/10.1128/MCB.00132-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580706PMC
July 2019

Distinctive structural basis for DNA recognition by the fission yeast Zn2Cys6 transcription factor Pho7 and its role in phosphate homeostasis.

Nucleic Acids Res 2018 11;46(21):11262-11273

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

Pho7, a member of the Zn2Cys6 family of fungal transcription factors, is the key transcriptional activator underlying fission yeast phosphate homeostasis, a physiological response to phosphate starvation in which the pho1, pho84 and tgp1 genes are upregulated. Here, we delineated a minimized 61-amino-acid Pho7 DNA-binding domain (DBD) and determined the 1.7 Å crystal structure of the DBD at its target site in the tgp1 promoter. Two distinctive features of the Pho7 DBD are: it binds DNA as a monomer, unlike most other fungal zinc-cluster factors that bind as homodimers; and it makes extensive interactions with its asymmetric target sequence over a 14-bp footprint that entails hydrogen bonding to 13 individual bases within, and remote from, the CGG triplet typically recognized by other Zn2Cys6 DBDs. Base pair substitutions at Pho7 sites in the tgp1 and pho1 promoters highlight the importance of the 5'-CGG triplet for Pho7 binding in vitro and Pho7-dependent gene expression in vivo. We identify several DBD amino acids at which alanine substitution effaced or attenuated the pho1 phosphate starvation response and concordantly reduced Pho7 binding to a pho1 promoter site.
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http://dx.doi.org/10.1093/nar/gky827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265462PMC
November 2018

A long noncoding (lnc)RNA governs expression of the phosphate transporter Pho84 in fission yeast and has cascading effects on the flanking lncRNA and genes.

J Biol Chem 2018 03 2;293(12):4456-4467. Epub 2018 Feb 2.

the Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York 10065

The expression of the phosphate transporter Pho84 in fission yeast is repressed in phosphate-rich medium and induced during phosphate starvation. Two other phosphate-responsive genes in ( and ) had been shown to be repressed in by transcription of a long noncoding (lnc) RNA from the upstream flanking gene, but whether expression is regulated in this manner is unclear. Here, we show that repression of is enforced by transcription of the SPBC8E4.02c locus upstream of to produce a lncRNA that we name ( -epressive ranscript 2). We identify two essential elements of the promoter, a HomolD box and a TATA box, mutations of which inactivate the promoter and de-repress the downstream promoter under phosphate-replete conditions. We find that promoter inactivation also elicits a cascade effect on the adjacent downstream (lncRNA) and (acid phosphatase) genes, whereby increased transcription down-regulates lncRNA transcription and thereby de-represses Our results establish a unified model for the repressive arm of fission yeast phosphate homeostasis, in which transcription of , , and lncRNAs interferes with the promoters of the flanking , , and genes, respectively.
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http://dx.doi.org/10.1074/jbc.RA117.001352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868275PMC
March 2018

Defining the DNA Binding Site Recognized by the Fission Yeast ZnCys Transcription Factor Pho7 and Its Role in Phosphate Homeostasis.

mBio 2017 08 15;8(4). Epub 2017 Aug 15.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA

Fission yeast phosphate homeostasis entails transcriptional induction of genes encoding phosphate-mobilizing proteins under conditions of phosphate starvation. Transcription factor Pho7, a member of the ZnCys family of fungal transcription regulators, is the central player in the starvation response. The DNA binding sites in the promoters of phosphate-responsive genes have not been defined, nor have any structure-function relationships been established for the Pho7 protein. Here we narrow this knowledge gap by (i) delineating an autonomous DNA-binding domain (DBD) within Pho7 that includes the ZnCys module, (ii) deploying recombinant Pho7 DBD in DNase I footprinting and electrophoretic mobility shift assays (EMSAs) to map the Pho7 recognition sites in the promoters of the phosphate-regulated and genes to a 12-nucleotide sequence motif [5'-TCG(G/C)(A/T)xxTTxAA], (iii) independently identifying the same motif as a Pho7 recognition element via analysis of available genome-wide ChIP-seq data, (iv) affirming that mutations in the two Pho7 recognition sites in the promoter efface expression , and (v) establishing that the zinc-binding cysteines and a pair of conserved arginines in the DBD are essential for Pho7 activity Fungi respond to phosphate starvation by inducing the transcription of a set of phosphate acquisition genes that comprise a phosphate regulon. Pho7, a member of the ZnCys family of fungal transcription regulators, is the central player in the phosphate starvation response in fission yeast. The present study identifies a 12-nucleotide Pho7 DNA binding motif [5'-TCG(G/C)(A/T)xxTTxAA] in the promoters of phosphate-regulated genes, pinpoints DNA and protein features important for Pho7 binding to DNA, and correlates them with Pho7-dependent gene expression The results highlight distinctive properties of Pho7 vis-a-vis other fungal zinc binuclear cluster transcription factors as well as the divergent cast of transcription factors deployed for phosphate homeostasis in fission yeast versus budding yeast.
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http://dx.doi.org/10.1128/mBio.01218-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559640PMC
August 2017

A new transcription factor for mitosis: in Schizosaccharomyces pombe, the RFX transcription factor Sak1 works with forkhead factors to regulate mitotic expression.

Nucleic Acids Res 2015 Aug 23;43(14):6874-88. Epub 2015 Apr 23.

Department of Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.

Mitotic genes are one of the most strongly oscillating groups of genes in the eukaryotic cell cycle. Understanding the regulation of mitotic gene expression is a key issue in cell cycle control but is poorly understood in most organisms. Here, we find a new mitotic transcription factor, Sak1, in the fission yeast Schizosaccharomyces pombe. Sak1 belongs to the RFX family of transcription factors, which have not previously been connected to cell cycle control. Sak1 binds upstream of mitotic genes in close proximity to Fkh2, a forkhead transcription factor previously implicated in regulation of mitotic genes. We show that Sak1 is the major activator of mitotic gene expression and also confirm the role of Fkh2 as the opposing repressor. Sep1, another forkhead transcription factor, is an activator for a small subset of mitotic genes involved in septation. From yeasts to humans, forkhead transcription factors are involved in mitotic gene expression and it will be interesting to see whether RFX transcription factors may also be involved in other organisms.
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http://dx.doi.org/10.1093/nar/gkv274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538799PMC
August 2015

Spectrophotometric estimation of functional groups on microslides for preparation of biochips.

Anal Biochem 2006 Apr 17;351(2):273-81. Epub 2006 Jan 17.

Nucleic Acids Research Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi University Campus, Delhi 110 007, India.

A universal reagent 1-O-(4,4'-dimethoxytrityl)-6-aminohexanol (DTAH) is described for the estimation of surface-bound functionalities (epoxy, aldehyde, and carboxyl) required for preparation of oligonucleotide arrays (biochips). The method involves the reaction of universal reagent DTAH with surface-bound functionality under microwaves for 10 min, followed by washings to remove the excess reagent. In the subsequent step, a weighed amount of DTAH-treated surface is exposed to acid to liberate 4,4'-dimethoxytrityl cation, which is measured at 505 nm to determine the functional group loading on the surface.
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http://dx.doi.org/10.1016/j.ab.2005.12.027DOI Listing
April 2006