Ribosomal Protein Gene Promoters Research Articles

The Product of the Fission Yeast fhl1 Gene Binds to the HomolE Box and Activates In Vitro Transcription of Ribosomal Protein Genes.

Fission yeast ribosomal protein genes (RPGs) contain a HomolD box as a core promoter element required for transcription. Some of the RPGs also contain a consensus sequence named HomolE, located upstream of the HomolD box. The HomolE box acts as an upstream activating sequence (UAS), and it is able to activate transcription in RPG promoters containing a HomolD box. In this work, we identified a HomolE-binding protein (HEBP) as a polypeptide of 100 kDa, which was able to bind to the HomolE box in a Southwestern blot assay. The features of this polypeptide were similar to the product of the fhl1 gene of fission yeast. The Fhl1 protein is the homolog of the FHL1 protein of budding yeast and possesses fork-head-associated (FHA) and fork-head (FH) domains. The product of the fhl1 gene was expressed and purified from bacteria, and it was demonstrated that is able to bind the HomolE box in an electrophoretic mobility assay (EMSA), as well as being able to activate in vitro transcription from an RPG gene promoter containing HomolE boxes upstream of the HomolD box. These results indicate that the product of the fhl1 gene of fission yeast can bind to the HomolE box, and it activates the transcription of RPGs.

ETS1 acts as a regulator of human healthy aging via decreasing ribosomal activity.

Adaptation to reduced energy production during aging is a fundamental issue for maintaining healthspan or prolonging life span. Currently, however, the underlying mechanism in long-lived people remains poorly understood. Here, we analyzed transcriptomes of 193 long-lived individuals (LLIs) and 86 spouses of their children from two independent Chinese longevity cohorts and found that the ribosome pathway was significantly down-regulated in LLIs. We found that the down-regulation is likely controlled by ETS1 (ETS proto-oncogene 1), a transcription factor down-regulated in LLIs and positively coexpressed with most ribosomal protein genes (RPGs). Functional assays showed that ETS1 can bind to RPG promoters, while ETS1 knockdown reduces RPG expression and alleviates cellular senescence in human dermal fibroblast (HDF) and embryonic lung fibroblast (IMR-90) cells. As protein synthesis/turnover in ribosomes is an energy-intensive cellular process, the decline in ribosomal biogenesis governed by ETS1 in certain female LLIs may serve as an alternative mechanism to achieve energy-saving and healthy aging.

Ribbon boosts ribosomal protein gene expression to coordinate organ form and function.

Cell growth is well defined for late (postembryonic) stages of development, but evidence for early (embryonic) cell growth during postmitotic morphogenesis is limited. Here, we report early cell growth as a key characteristic of tubulogenesis in the Drosophila embryonic salivary gland (SG) and trachea. A BTB/POZ domain nuclear factor, Ribbon (Rib), mediates this early cell growth. Rib binds the transcription start site of nearly every SG-expressed ribosomal protein gene (RPG) and is required for full expression of all RPGs tested. Rib binding to RPG promoters in vitro is weak and not sequence specific, suggesting that specificity is achieved through cofactor interactions. Accordingly, we demonstrate Rib's ability to physically interact with each of the three known regulators of RPG transcription. Surprisingly, Rib-dependent early cell growth in another tubular organ, the embryonic trachea, is not mediated by direct RPG transcription. These findings support a model of early cell growth customized by transcriptional regulatory networks to coordinate organ form and function.

Transcriptional control of ribosome biogenesis in yeast: links to growth and stress signals.

Ribosome biogenesis requires prodigious transcriptional output in rapidly growing yeast cells and is highly regulated in response to both growth and stress signals. This minireview focuses on recent developments in our understanding of this regulatory process, with an emphasis on the 138 ribosomal protein genes (RPGs) themselves and a group of >200 ribosome biogenesis (RiBi) genes whose products contribute to assembly but are not part of the ribosome. Expression of most RPGs depends upon Rap1, a pioneer transcription factor (TF) required for the binding of a pair of RPG-specific TFs called Fhl1 and Ifh1. RPG expression is correlated with Ifh1 promoter binding, whereas Rap1 and Fhl1 remain promoter-associated upon stress-induced down regulation. A TF called Sfp1 has also been implicated in RPG regulation, though recent work reveals that its primary function is in activation of RiBi and other growth-related genes. Sfp1 plays an important regulatory role at a small number of RPGs where Rap1–Fhl1–Ifh1 action is subsidiary or non-existent. In addition, nearly half of all RPGs are bound by Hmo1, which either stabilizes or re-configures Fhl1–Ifh1 binding. Recent studies identified the proline rotamase Fpr1, known primarily for its role in rapamycin-mediated inhibition of the TORC1 kinase, as an additional TF at RPG promoters. Fpr1 also affects Fhl1–Ifh1 binding, either independently or in cooperation with Hmo1. Finally, a major recent development was the discovery of a protein homeostasis mechanism driven by unassembled ribosomal proteins, referred to as the Ribosome Assembly Stress Response (RASTR), that controls RPG transcription through the reversible condensation of Ifh1.

Physiological function of FKBP12, a primary target of rapamycin/FK506: a newly identified role in transcription of ribosomal protein genes in yeast.

In this review, we have summarized the information from a study on FKBP12 (FK506 binding protein 12kDa) with a view to understand its drug-free, physiological roles in transcription of ribosomal protein gene in Saccharomyces cerevisiae. FKBP12 with peptidyl-prolylisomerase (PPIase) activity is widely conserved among many eukaryotes. FKBP12 is a primary target for the two structurally related drugs, FK506 and rapamycin. FKBP12 bound with FK506 or rapamycin inhibits calcineurin and target of rapamycin complex 1 (TORC1), respectively. The molecular mechanisms of the effect of FKBP12 in the presence of these drugs have been elucidated. Conversely, the physiological role of FKBP12 has been unclear, especially in yeast. Our study revealed that the deletion of FPR1 (FK506-sensitive prolinerotamase 1 gene), which encodes yeast FKBP12, induced severe growth defect synthetically with deletion of HMO1 (high mobility group family 1). HMO1 encodes an HMGB family protein involved in transcription of ribosomal component genes. Fpr1 was shown to bind specifically to the promoters of ribosomal protein genes (RPGs) dependent on Rap1 (repressor/activator binding protein 1). Importantly, Fpr1 and Hmo1 promote the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), key regulators of RPG transcription, to certain RPG promoters independently and/or cooperatively with each other. Taken together, we conclude that Fpr1 physiologically functions as transcription factor of RPGs in S. cerevisiae. To our knowledge, this is the first study to demonstrate that FKBP12 participates in ribosome synthesis independently of drugs, and it may also provide a clue to the unidentified function of other PPIase proteins.

Mechanisms coordinating ribosomal protein gene transcription in response to stress.

While expression of ribosomal protein genes (RPGs) in the budding yeast has been extensively studied, a longstanding enigma persists regarding their co-regulation under fluctuating growth conditions. Most RPG promoters display one of two distinct arrangements of a core set of transcription factors (TFs) and are further differentiated by the presence or absence of the HMGB protein Hmo1. However, a third group of promoters appears not to be bound by any of these proteins, raising the question of how the whole suite of genes is co-regulated. We demonstrate here that all RPGs are regulated by two distinct, but complementary mechanisms driven by the TFs Ifh1 and Sfp1, both of which are required for maximal expression in optimal conditions and coordinated downregulation upon stress. At the majority of RPG promoters, Ifh1-dependent regulation predominates, whereas Sfp1 plays the major role at all other genes. We also uncovered an unexpected protein homeostasis-dependent binding property of Hmo1 at RPG promoters. Finally, we show that the Ifh1 paralog Crf1, previously described as a transcriptional repressor, can act as a constitutive RPG activator. Our study provides a more complete picture of RPG regulation and may serve as a paradigm for unravelling RPG regulation in multicellular eukaryotes.

Fpr1, a primary target of rapamycin, functions as a transcription factor for ribosomal protein genescooperatively with Hmo1 in Saccharomyces cerevisiae.

Fpr1 (FK506-sensitive proline rotamase 1), a protein of the FKBP12 (FK506-binding protein 12 kDa) family in Saccharomyces cerevisiae, is a primary target for the immunosuppressive agents FK506 and rapamycin. Fpr1 inhibits calcineurin and TORC1 (target of rapamycin complex 1) when bound to FK506 and rapamycin, respectively. Although Fpr1 is recognised to play a crucial role in the efficacy of these drugs, its physiological functions remain unclear. In a hmo1Δ (high mobility group family 1-deleted) yeast strain, deletion of FPR1 induced severe growth defects, which could be alleviated by increasing the copy number of RPL25 (ribosome protein of the large subunit 25), suggesting that RPL25 expression was affected in hmo1Δfpr1Δ cells. In the current study, extensive chromatin immunoprecipitation (ChIP) and ChIP-sequencing analyses revealed that Fpr1 associates specifically with the upstream activating sequences of nearly all RPG (ribosomal protein gene) promoters, presumably in a manner dependent on Rap1 (repressor/activator site binding protein 1). Intriguingly, Fpr1 promotes the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), two key regulators of RPG transcription, to certain RPG promoters independently of and/or cooperatively with Hmo1. Furthermore, mutation analyses of Fpr1 indicated that for transcriptional function on RPG promoters, Fpr1 requires its N-terminal domain and the binding surface for rapamycin, but not peptidyl-prolyl isomerase activity. Notably, Fpr1 orthologues from other species also inhibit TORC1 when bound to rapamycin, but do not regulate transcription in yeast, which suggests that these two functions of Fpr1 are independent of each other.

Shared distal regulatory regions may contribute to the coordinated expression of human ribosomal protein genes

To identify the potential distal regulatory regions of human ribosomal protein genes (RPGs) and to understand their characteristics, we studied the chromatin interactions in seven cell lines and four primary cell types. We identified 22,797 putative regulatory regions that directly or indirectly interact with human RPG promoters. A large proportion of these regions are only present in one cell line or one cell type, implying that RPGs may be differentially regulated across experimental conditions. We also noticed that groups of RPGs, which are the same groups across cell lines and cell types, share common regulatory regions. These shared regulatory regions by RPGs may contribute to their coordinated regulation. By studying the overrepresented motifs in the identified regulatory regions, we showed that there are about two dozen motifs in these regions shared across cell lines and cell types. Our study shed new light on the coordinated transcriptional regulation of human RPGs.

A ribosome assembly stress response regulates transcription to maintain proteome homeostasis.

Ribosome biogenesis is a complex and energy-demanding process requiring tight coordination of ribosomal RNA (rRNA) and ribosomal protein (RP) production. Given the extremely high level of RP synthesis in rapidly growing cells, alteration of any step in the ribosome assembly process may impact growth by leading to proteotoxic stress. Although the transcription factor Hsf1 has emerged as a central regulator of proteostasis, how its activity is coordinated with ribosome biogenesis is unknown. Here, we show that arrest of ribosome biogenesis in the budding yeast Saccharomyces cerevisiae triggers rapid activation of a highly specific stress pathway that coordinately upregulates Hsf1 target genes and downregulates RP genes. Activation of Hsf1 target genes requires neo-synthesis of RPs, which accumulate in an insoluble fraction and presumably titrate a negative regulator of Hsf1, the Hsp70 chaperone. RP aggregation is also coincident with that of the RP gene activator Ifh1, a transcription factor that is rapidly released from RP gene promoters. Our data support a model in which the levels of newly synthetized RPs, imported into the nucleus but not yet assembled into ribosomes, work to continuously balance Hsf1 and Ifh1 activity, thus guarding against proteotoxic stress during ribosome assembly.

Distinct core promoter codes drive transcription initiation at key developmental transitions in a marine chordate

BackgroundDevelopment is largely driven by transitions between transcriptional programs. The initiation of transcription at appropriate sites in the genome is a key component of this and yet few rules governing selection are known. Here, we used cap analysis of gene expression (CAGE) to generate bp-resolution maps of transcription start sites (TSSs) across the genome of Oikopleura dioica, a member of the closest living relatives to vertebrates.ResultsOur TSS maps revealed promoter features in common with vertebrates, as well as striking differences, and uncovered key roles for core promoter elements in the regulation of development. During spermatogenesis there is a genome-wide shift in mode of transcription initiation characterized by a novel core promoter element. This element was associated with > 70% of male-specific transcription, including the use of cryptic internal promoters within operons. In many cases this led to the exclusion of trans-splice sites, revealing a novel mechanism for regulating which mRNAs receive the spliced leader. Binding of the cell cycle regulator, E2F1, is enriched at the TSS of maternal genes in endocycling nurse nuclei. In addition, maternal promoters lack the TATA-like element found in zebrafish and have broad, rather than sharp, architectures with ordered nucleosomes. Promoters of ribosomal protein genes lack the highly conserved TCT initiator. We also report an association between DNA methylation on transcribed gene bodies and the TATA-box.ConclusionsOur results reveal that distinct functional promoter classes and overlapping promoter codes are present in protochordates like in vertebrates, but show extraordinary lineage-specific innovations. Furthermore, we uncover a genome-wide, developmental stage-specific shift in the mode of TSS selection. Our results provide a rich resource for the study of promoter structure and evolution in Metazoa.

Opbp is a new architectural/insulator protein required for ribosomal gene expression.

A special class of poorly characterized architectural proteins is required for chromatin topology and enhancer–promoter interactions. Here, we identify Opbp as a new Drosophila architectural protein, interacting with CP190 both in vivo and in vitro. Opbp binds to a very restrictive set of genomic regions, through a rare sequence specific motif. These sites are co-bound by CP190 in vivo, and generally located at bidirectional promoters of ribosomal protein genes. We show that Opbp is essential for viability, and loss of opbp function, or destruction of its motif, leads to reduced ribosomal protein gene expression, indicating a functional role in promoter activation. As characteristic of architectural/insulator proteins, the Opbp motif is sufficient for distance-dependent reporter gene activation and enhancer-blocking activity, suggesting an Opbp-mediated enhancer–promoter interaction. Rather than having a constitutive role, Opbp represents a new type of architectural protein with a very restricted, yet essential, function in regulation of housekeeping gene expression.

A sequence-specific core promoter-binding transcription factor recruits TRF2 to coordinately transcribe ribosomal protein genes.

Ribosomal protein (RP) genes must be coordinately expressed for proper assembly of the ribosome yet the mechanisms that control expression of RP genes in metazoans are poorly understood. Recently, TATA-binding protein-related factor 2 (TRF2) rather than the TATA-binding protein (TBP) was found to function in transcription of RP genes in Drosophila. Unlike TBP, TRF2 lacks sequence-specific DNA binding activity, so the mechanism by which TRF2 is recruited to promoters is unclear. We show that the transcription factor M1BP, which associates with the core promoter region, activates transcription of RP genes. Moreover, M1BP directly interacts with TRF2 to recruit it to the RP gene promoter. High resolution ChIP-exo was used to analyze in vivo the association of M1BP, TRF2 and TFIID subunit, TAF1. Despite recent work suggesting that TFIID does not associate with RP genes in Drosophila, we find that TAF1 is present at RP gene promoters and that its interaction might also be directed by M1BP. Although M1BP associates with thousands of genes, its colocalization with TRF2 is largely restricted to RP genes, suggesting that this combination is key to coordinately regulating transcription of the majority of RP genes in Drosophila.

A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis

Cell growth potential is determined by the rate ofribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. Inthe yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription.

Role of CK2-dependent phosphorylation of Ifh1 and Crf1 in transcriptional regulation of ribosomal protein genes in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Fhl1 is involved in the regulation of ribosomal protein (RP) genes through interaction with either its coactivator Ifh1 or corepressor Crf1, depending on nutrient conditions. Interaction of Fhl1 with Ifh1 or Crf1 is achieved through a forkhead-associated (FHA) domain of Fhl1, which binds to forkhead-binding (FHB) domains of Ifh1 and Crf1. Here, we demonstrate that CK2-dependent phosphorylation of T681 and T348 residues, located in the FHB domains of Ifh1 and Crf1, respectively, provides binding sites for the FHA domain of Fhl1. Cells expressing Ifh1T681A mutant showed reduced association of Ifh1 at the RP gene promoters and decreased levels of RP gene transcripts, thereby reducing the growth rate. On the other hand, cells expressing Crf1T348A showed a defect in repressing RP gene transcription upon inhibition of target of rapamycin complex 1 (TORC1) by rapamycin treatment. Taken together, these findings suggest the mechanisms by which CK2-dependent recruitment of Ifh1 and Crf1 at the RP gene promoters governs the transcription of RP genes.

Genome-wide identification and characterization of transcription start sites and promoters in the tunicate Ciona intestinalis.

The tunicate Ciona intestinalis, an invertebrate chordate, has recently emerged as a powerful model organism for gene regulation analysis. However, few studies have been conducted to identify and characterize its transcription start sites (TSSs) and promoters at the genome-wide level. Here, using TSS-seq, we identified TSSs at the genome-wide scale and characterized promoters in C. intestinalis. Specifically, we identified TSS clusters (TSCs), high-density regions of TSS-seq tags, each of which appears to originate from an identical promoter. TSCs were found not only at known TSSs but also in other regions, suggesting the existence of many unknown transcription units in the genome. We also identified candidate promoters of 79 ribosomal protein (RP) genes, each of which had the major TSS in a polypyrimidine tract and showed a sharp TSS distribution like human RP gene promoters. Ciona RP gene promoters, however, did not appear to have typical TATA boxes, unlike human RP gene promoters. In Ciona non-RP promoters, two pyrimidine-purine dinucleotides, CA and TA, were frequently used as TSSs. Despite the absence of CpG islands, Ciona TATA-less promoters showed low expression specificity like CpG-associated human TATA-less promoters. By using TSS-seq, we also predicted trans-spliced gene TSSs and found that their downstream regions had higher G+T content than those of non-trans-spliced gene TSSs. Furthermore, we identified many putative alternative promoters, some of which were regulated in a tissue-specific manner. Our results provide valuable information about TSSs and promoter characteristics in C. intestinalis and will be helpful in future analysis of transcriptional regulation in chordates.

Eaf1p Is Required for Recruitment of NuA4 in Targeting TFIID to the Promoters of the Ribosomal Protein Genes for Transcriptional Initiation In Vivo.

NuA4 (nucleosome acetyltransferase of H4) promotes transcriptional initiation of TFIID (a complex of TBP and TBP-associated factors [TAFs])-dependent ribosomal protein genes involved in ribosome biogenesis. However, it is not clearly understood how NuA4 regulates the transcription of ribosomal protein genes. Here, we show that NuA4 is recruited to the promoters of ribosomal protein genes, such as RPS5, RPL2B, and RPS11B, for TFIID recruitment to initiate transcription, and the recruitment of NuA4 to these promoters is impaired in the absence of its Eaf1p component. Intriguingly, impaired NuA4 recruitment in a Δeaf1 strain depletes recruitment of TFIID (a TAF-dependent form of TBP) but not the TAF-independent form of TBP to the promoters of ribosomal protein genes. However, in the absence of NuA4, SAGA (Spt-Ada-Gcn5-acetyltransferase) is involved in targeting the TAF-independent form of TBP to the promoters of ribosomal protein genes for transcriptional initiation. Thus, NuA4 plays an important role in targeting TFIID to the promoters of ribosomal protein genes for transcriptional initiation in vivo. Such a function is mediated via its targeted histone acetyltransferase activity. In the absence of NuA4, ribosomal protein genes lose TFIID dependency and become SAGA dependent for transcriptional initiation. Collectively, these results provide significant insights into the regulation of ribosomal protein gene expression and, hence, ribosome biogenesis and functions.

TOR and Proteasomal Regulation of Ribosomal Protein Gene Expression

Previous studies have implicated SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (Transcription factor-IID) dependent mechanisms of transcriptional activation in yeast. SAGA-dependent transcriptional activation is further regulated by the 19S proteasome subcomplex. However, the role of the 19S proteasome subcomplex in transcriptional activation of the TFIID-dependent genes has not been elucidated. Ribosomal protein (RP) genes are important class of TFIID-dependent genes, and are crucial for ribosome biogenesis, protein synthesis, and hence cellular growth and proliferation. Since RP genes are TFIID-dependent and the role of the 19S proteasome subcomplex in their transcription remain unknown, we investigated whether 19S proteasome subcomplex regulates the transcription of RP genes. We find that the 19S proteasome subcomplex is recruited to the promoters of RP genes and enhances the targeting of co-activator NuA4 HAT (histone acetyl transferase) to activator Rap1. Such enhanced targeting of NuA4 HAT is essential to bring down TFIID complex in a HAT-dependent manner. Intriguingly, the HAT activity of NuA4 complex is regulated by environmental factors such as heat, nutrition and osmotic pressure via TOR (Target of Rapamycin) signaling pathway. We also find that TOR facilitates the targeting of 19S proteasome subcomplex to the promoters of RP genes to promote NuA4 HAT association, histone H4 acetylation and the formation of pre-initiation complex at their promoters. Together, our results delineate new regulatory pathway of RP gene expression via TOR, 19S proteasome subcomplex and NuA4 HAT.

The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing.

Early steps of gene expression are a composite of promoter recognition, promoter activation, RNA synthesis and RNA processing, and it is known that SUMOylation, a post-translational modification, is involved in transcription regulation. We previously found that SUMO-1 marks chromatin at the proximal promoter regions of some of the most active housekeeping genes during interphase in human cells, but the SUMOylated targets on the chromatin remained unclear. In this study, we found that SUMO-1 marks the promoters of ribosomal protein genes via modification of the Scaffold Associated Factor B (SAFB) protein, and the SUMOylated SAFB stimulated both the binding of RNA polymerase to promoters and pre-mRNA splicing. Depletion of SAFB decreased RNA polymerase II binding to promoters and nuclear processing of the mRNA, though mRNA stability was not affected. This study reveals an unexpected role of SUMO-1 and SAFB in the stimulatory coupling of promoter binding, transcription initiation and RNA processing.

Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes.

Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs.

Both HMG boxes in Hmo1 are essential for DNA binding in vitro and in vivo.

Hmo1, a member of the high mobility group B family proteins in Saccharomyces cerevisiae, associates with the promoters of ribosomal protein genes (RPGs) to direct accurate transcriptional initiation. Here, to identify factors involved in the binding of Hmo1 to its targets and the mechanism of Hmo1-dependent transcriptional initiation, we developed a novel reporter system using the promoter of the RPG RPS5. A genetic screen did not identify any factors that influence Hmo1 binding, but did identify a number of mutations in Hmo1 that impair its DNA binding activity in vivo and in vitro. These results suggest that Hmo1 binds to its target promoters autonomously without any aid of additional factors. Furthermore, characterization of Hmo1 mutants showed that the box A domain plays a pivotal role in DNA binding and may be required for the recognition of structural properties of target promoters that occur in native chromatin.