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Transcriptional and Translational Regulatory Mechanisms in Nutrient Control of Gene Expression

• Alan G. Hinnebusch, PhD, Head, Section on Nutrient Control of Gene Expression
• Hongfang Qiu, PhD, Staff Scientist
• Fan Zhang, MS, Senior Research Assistant
• Swati Gaikwad, PhD, Postdoctoral Fellow
• Ritu Gupta, PhD, Postdoctoral Fellow
• Rakesh Kumar, PhD, Postdoctoral Fellow
• Priyanka Mittal, PhD, Postdoctoral Fellow
• Poonam Poonia, PhD, Postdoctoral Fellow
• Priyanka Singh, PhD, Postdoctoral Fellow
• Sakshi Singh, PhD, Postdoctoral Fellow
• Vishalini Valabhoju, PhD, Postdoctoral Fellow
• Anil Vijjamarri, PhD, Postdoctoral Fellow

We study the fundamental mechanisms involved in the assembly and function of translation initiation complexes for protein synthesis, using yeast as a model system in order to exploit its powerful combination of genetics and biochemistry. The translation initiation pathway produces an 80S ribosome bound to mRNA, with methionyl initiator tRNA (Met-tRNA_i) base-paired to the AUG start codon. The Met-tRNA_i is recruited to the small (40S) subunit in a ternary complex (TC) with the GTP–bound eukaryotic initiation factor eIF2 to produce the 43S preinitiation complex (PIC) in a reaction stimulated by eIFs 1, 1A, 3, and 5. The 43S PIC attaches to the 5′ end of mRNA, facilitated by the cap-binding complex eIF4F (comprising eIF4E, eIF4G, and the RNA helicase eIF4A) and poly(A)–binding protein (PABP) bound to the poly(A) tail, and scans the 5′ untranslated region (UTR) for the AUG start codon. Scanning is promoted by eIF1 and eIF1A, which induce an open conformation of the 40S and rapid TC binding in a conformation suitable for the scanning of successive triplets entering the ribosomal P site (P-out), and by eIF4F and other RNA helicases, such as Ded1 and its paralog Dbp1, that remove secondary structure in the 5′ UTR. AUG recognition evokes tighter binding of the TC in the P-in state and irreversible GTP hydrolysis by eIF2, dependent on the GTPase–activating protein (GAP) eIF5, releasing eIF2-GDP from the PIC, with Met-tRNA_i remaining in the P site. Joining of the 60S subunit produces the 80S initiation complex ready for protein synthesis.

Our current aims in this research area are to: (1) elucidate the functions of eIF1, eIF5, eIF3, and 40S ribosomal proteins in TC recruitment and start-codon recognition; (2) identify distinct functions of the RNA helicases eIF4A (and its cofactors eIF4G/eIF4B), Ded1, and Dbp1, and of the poly(A)–binding protein (PABP) in mRNA activation, 48S PIC assembly, and scanning in vivo; (3) uncover the mechanisms of translational repression and regulation of mRNA abundance by the repressors Scd6, Pat1, the helicase Dhh1, and the mRNA–decapping enzyme Dcp2, and identify the RNA–binding proteins involved in these functions; (4) elucidate the regulation of Ded1, eIF4G, and Dhh1 functions in response to nutrient limitation or stress; (5) elucidate the roles of the yeast orthologs of eIF2A and eIF2D in eIF2–independent initiation of translation in stress conditions; and (6) elucidate the role of eIF4E–binding protein Eap1 in regulating mRNA decay and translation.

We also analyze the regulation of amino acid–biosynthetic genes in budding yeast as a means to dissect fundamental mechanisms of transcriptional control of gene expression. During amino acid limitation, transcription of such genes is coordinately induced by the activator Gcn4 as the result of its induction at the translational level. The eviction of nucleosomes that occlude promoter DNA sequences and block access by RNA polymerase is thought to be a rate-limiting step for transcriptional activation. Previous studies implicated certain histone chaperones, ATP–dependent chromatin-remodeling complexes, or histone acetyltransferase (HAT) complexes in eviction of promoter nucleosomes at certain yeast genes, but it is unclear whether these co-factors function at Gcn4 target genes. Our aim is to elucidate the full set of co-factors that participate in promoter nucleosome eviction and preinitiation complex (PIC) assembly at Gcn4 target genes, their involvement in this process genome-wide, and the transcriptional consequences of defective nucleosome eviction or recruitment of the general transcription factor TATA–binding protein (TBP). Functional cooperation among the chromatin-remodeling complexes SWI/SNF, RSC, and Ino80, as well as the HAT complexes SAGA, NuA4, NuA3, and Rtt109/Asf1, in these processes has been identified. We recently discovered that Gcn4 can activate transcription from binding sites within the coding sequences (CDS) of its target genes, inducing internal subgenic sense and antisense (AS) transcripts in addition to the conventional full-length transcripts that initiate 5′ of the CDS; and we are probing both the mechanism and possible regulatory functions of these internal AS transcripts, as well as the roles of co-transcriptional histone methylation, nucleosome reassembly, and mRNA decay enzymes in controlling their synthesis and abundance. We are also probing mechanisms involved in the asymmetric transcriptional induction of genes belonging to pairs of divergently oriented genes where only one gene responds to Gcn4 binding at the shared upstream activation sequences (enhancer); and the relative contributions of co-factors SAGA, TFIID, and Mot1 to TBP recruitment.

Differential requirements for P-stalk components in activating yeast protein kinase Gcn2 by stalled ribosomes during stress

A highly conserved response to amino acid starvation involves activation of the protein kinase Gcn2, which phosphorylates eukaryotic initiation factor 2, with attendant inhibition of global protein synthesis and increased translation of the yeast transcriptional activator GCN4. Gcn2 contains a domain related to histidyl-tRNA synthetase (HisRS–like domain), and a C-terminal ribosome-binding domain. Previous work indicated that Gcn2 is activated on translating ribosomes by uncharged tRNAs that accumulate in amino acid–starved cells and pair with the cognate codons in the empty ribosomal A, interacting directly with the HisRS–like domain to stimulate kinase activity. Gcn2 can also be activated by conditions that stall elongating ribosomes without reducing aminoacylation of tRNA, but it was unclear whether distinct molecular mechanisms operate in these two circumstances. We identified three regimes that activate Gcn2 in yeast by starvation-independent (SI) ribosome stalling, which leaves an empty A site on the stalled ribosome: (1) treatment with inhibitor tigecycline, which should stall ribosomes at all codons; (2) deleting the gene encoding tRNAArgUCC, which should stall ribosomes at AGG codons; and (3) depletion of translation-termination factor eRF1, which should stall ribosomes at stop codons. Subsequent genetic analysis demonstrated requirements for the HisRS–like and ribosome-binding domains of Gcn2, positive effectors Gcn1/Gcn20, and the tethering of at least one of two P1/P2 heterodimers of the 60S ribosomal P-stalk complex, for detectable activation by SI–ribosome stalling. Remarkably, no tethered P1/P2 proteins were required for strong Gcn2 activation by starvation for various amino acids, indicating that Gcn2 activation has different requirements for the P-stalk, depending on how ribosomes are stalled. We propose that accumulation of deacylated tRNAs in starved cells functionally substitutes for the P-stalk in binding to the HisRS–like domain for eIF2 kinase activation by ribosomes stalled with A sites.

Yeast mRNA decapping factors control mRNA abundance and translation to adjust metabolism and cell filamentation to nutrient availability.

Pat1 and helicase Dhh1 are conserved activators of the mRNA decapping enzyme Dcp1:Dcp2, are central to general mRNA decay, and were implicated in repressing translation in glucose-starved cells. Using ribosome profiling and RNA-seq analysis of dhh1, pat1, and dhh1pat1 mutants cultured in rich medium, we identified hundreds of mRNAs up-regulated in a manner indicating cumulative repression by Pat1 and Dhh1. Although the environmental stress response (ESR) is mobilized in these mutants, involving increased expression of stress genes (iESR) and repression of ribosome production and translation factors (rESR), most up-regulated mRNAs are not iESR transcripts. CAGE (cap analysis of gene expression) analyses of capped mRNAs revealed enhanced accumulation of decapped intermediates for the up-regulated transcripts, and ChIP-seq analysis of RNA Pol II indicated decreased rather than increased transcription of the cognate genes, demonstrating that reduced decapping and 5′-3′ pathway degradation drives transcript derepression in the mutants. The cumulative contributions of Dhh1 and Pat1 to mRNA decapping are consistent with their independent interactions with distinct segments of Dcp2 involved in its activation, and evidence for distinct decapping complexes containing Dhh1 or Pat1. Although previous work implicated Dhh1 and Pat1 in accelerating degradation of mRNAs enriched for slowly decoded codons, the mRNAs up-regulated in the mutants have average proportions of suboptimal codons. Pat1 and Dhh1 also collaborate to reduce the translational efficiencies (TEs) and protein production of many mRNAs, including highly repressed mRNAs involved in cell adhesion or utilization of the poor nitrogen source allantoin. Pat1/Dhh1 also repress the abundance or TE of transcripts involved in oxidative phosphorylation (OXPHOS), catabolism of non-preferred carbon or nitrogen sources, or in autophagy. We obtained evidence for increased activity of the electron transport chain (ETC) of OXPHOS in the dhh1 and pat1 mutants, and elevated autophagic flux in the pat1/dhh1 double mutant. As these genes/pathways are normally repressed in cells growing in rich medium replete, we concluded that Pat1 and Dhh1 function as post-transcriptional repressors of multiple pathways normally activated only during nutrient limitation.

Parallel analysis of the dcp2 mutant led to the finding that, among the 1,300 mRNAs preferentially targeted for degradation by the decapping enzyme, 55% utilize Dhh1/Pat1 or decapping activators Scd6/Edc3 to promote decay, while the remainder employ the Upf factors that mediate nonsense-mediated mRNA decay (NMD). We also found that the dcp2 mutation confers a broad reprogramming of translation, wherein well translated mRNAs exhibit increased TEs at the expense of poorly translated mRNAs, which we could attribute to increased competition for 43S PICs, given that dcp2 cells contain elevated mRNA levels coupled with reduced ribosome abundance (owing to the ESR response). The increased mRNA/40S ratio and decreased 40S concentration should favor mRNAs with high rates of PIC recruitment at the expense of poorly initiated transcripts. As might be expected, the dcp2 mutation up-regulates many of the same mRNAs required for respiration, utilization of poor carbon/nitrogen sources, and autophagy derepressed in the pat1 and dhh1 mutants, and confers elevated mitochondrial membrane potential and TCA cycle intermediates, indicating increased OXPHOS on glucose-rich medium. The dcp2 mutant cells also resemble the decapping activator mutants in showing elevated expression of cell-adhesion proteins that function in forming pseudohyphae, and we observed increased filamentation of both dcp2 and pat1 mutant cells on rich medium. As filamentation is normally limited to starvation conditions and is viewed as a strategy for nutrient foraging, this phenotype supports the role of decapping factors in repressing pathways utilized primarily in starved cells.

Distinct functions of three chromatin remodelers in activator binding and preinitiation-complex (PIC) assembly

The nucleosome-remodeling complexes (CRs) SWI/SNF, RSC, and Ino80C cooperate in evicting or repositioning nucleosomes to produce nucleosome-depleted regions (NDRs) at the promoters of many yeast genes induced by amino acid starvation. We analyzed mutants lacking the CR catalytic subunits for binding of the transcriptional activator Gcn4 and recruitment of TATA–binding protein (TBP) during PIC assembly. RSC and Ino80 enhance Gcn4 binding to UAS (upstream activation sequence) elements in NDRs upstream of many promoters, as well as to unconventional binding sites within nucleosome-occupied coding sequences; and SWI/SNF contributes to UAS binding when RSC is depleted. All three CRs are actively recruited by Gcn4 to most UAS elements and appear to enhance Gcn4 binding by reducing nucleosome occupancies at the binding motifs, indicating a positive regulatory loop. SWI/SNF acts unexpectedly in wild-type cells to prevent excessive Gcn4 binding at certain UAS elements, which might involve transient nucleosome sliding that does not alter steady-state nucleosome occupancies. All three CRs also stimulate TBP recruitment, at least partly by reducing nucleosome occupancies at TBP binding sites, with SWI/SNF acting preferentially at the most highly expressed Gcn4 target genes. RSC and Ino80 function more broadly than SWI/SNF to stimulate TBP recruitment at most constitutively expressed genes, including ribosomal protein genes, whereas SWI/SNF acts preferentially at a distinct subset of highly expressed genes. Our findings point to a complex interplay among the three CRs in evicting promoter nucleosomes to regulate activator binding and stimulate PIC assembly.

Differential requirements for Gcn5 and NuA4 HAT activities in the starvation-induced versus basal transcriptomes

Previously, we showed that elimination of Gcn5, the histone acetyltransferase (HAT) subunit in co-factor SAGA, did not fully impair nucleosome eviction at many starvation-induced genes, suggesting that Gcn5 might cooperate with other HATs in this process, similar to the functional cooperation we had identified for different CRs. The role of the HAT complex NuA4, responsible for most H4 and H2A acetylation in yeast, was of particular interest, as it was shown to be recruited to the Gcn4 target genes ARG1 and ARG4. We examined the effects of disrupting the NuA4 complex, by eliminating its nonessential scaffold subunit Eaf1, on promoter nucleosome eviction and transcriptional activation at both starvation-induced and constitutively expressed genes. We also examined whether depleting Eaf1 from the nucleus (by anchor-away technology) confers defects in nucleosome eviction or transcription in cells lacking Gcn5 in order to evaluate whether NuA4 and Gcn5 make independent, additive contributions to these processes at particular genes in vivo. Our results revealed that NuA4 acts on par with Gcn5, and functions additively, in evicting and repositioning promoter nucleosomes, and in stimulating transcription, at starvation-induced genes. However, NuA4 is generally more important than Gcn5 in promoter nucleosome eviction, recruitment of the TATA-binding protein (TBP), and transcription at most other genes expressed constitutively in yeast. NuA4 also predominates over Gcn5 in stimulating TBP recruitment and transcription of genes categorized as principally dependent on the cofactor TFIID versus SAGA, except for the highly expressed subset encoding ribosomal proteins (RPs), where Gcn5 contributes strongly to PIC assembly and transcription. We found that both SAGA and NuA4 are recruited to promoter regions of starvation-induced genes in a manner that appears to be controlled by their HAT activities, and thus most likely act directly to promote transcription of these genes. Our findings reveal an intricate interplay between these two HATs in nucleosome eviction, PIC assembly, and transcription that differs between the starvation-induced and basal transcriptomes.

Publications

1. Gupta R, Hinnebusch AG. Differential requirements for P-stalk components in activating yeast protein kinase Gcn2 by stalled ribosomes during stress. Proc Natl Acad Sci USA 2023 120:e2300521120.
2. Vijjamarri AK, Niu X, Vandermeulen MD, Onu C, Zhang F, Qiu H, Gupta N, Gaikwad S, Greenberg ML, Cullen PJ, Lin Z, Hinnebusch AG. Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability. eLife 2023 12:e85545.
3. Vijjamarri AK, Gupta N, Onu C, Niu X, Zhang F, Kumar R, Lin Z, Greenberg ML, Hinnebusch AG. mRNA decapping activators Pat1 and Dhh1 regulate transcript abundance and translation to tune cellular responses to nutrient availability. Nucleic Acids Res 2023 51:9314–9336.
4. Rawal Y, Qiu H, Hinnebusch AG. Distinct functions of three chromatin remodelers in activator binding and preinitiation complex assembly. PLoS Genet 2022 18:e1010277.
5. Zheng Q, Qiu H, Zhang H, Hinnebusch AG. Differential requirements for Gcn5 and NuA4 HAT activities in the starvation-induced versus basal transcriptomes. Nucleic Acids Res 2023 51:3696–3721.
6. Dever TE, Ivanov IP, Hinnebusch AG. Translational regulation by uORFs and start codon selection stringency. Genes Dev 2023 37:474–489.

Collaborators

• Paul Cullen, PhD, Department of Biological Sciences, State University of Buffalo, Buffalo, NY
• Miriam L. Greenberg, PhD, Department of Biological Sciences, Wayne State University, Detroit, MI
• Zhenguo Lin, PhD, Department of Biology, Saint Louis University, St. Louis, MO
• Jose L. Llácer, PhD, Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain
• Jon Lorsch, PhD, Laboratory on the Mechanism and Regulation of Protein Synthesis, NICHD, Bethesda, MD
• Neelam Sen, PhD, Jawaharlal Nehru University, New Delhi, India

Contact

For more information, email hinnebua@mail.nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/hinnebusch.

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