Yeast Rad51 Research Articles

Abstract 229: A cross-species approach to identifying genetic modifiers of BRCA1-associated breast cancer risk

Abstract Background: Although carriers of germline mutations of BRCA1 have a high risk of developing breast and ovarian cancers, penetrance is not complete, and it is likely that unidentified genes modify the risk for individual mutation carriers. We hypothesize that genes that modify the penetrance of cancer in BRCA1 carriers interact with BRCA1 mutations to confer increased sensitivity to DNA damage at the cellular level, and that cellular sensitivity to DNA damage can be used as an intermediate phenotype to predict breast cancer susceptibility in humans. Objective: Our goal is to test a cross-species systems biology approach to predict gene-gene interactions underlying breast cancer susceptibility, using sensitivity to DNA damage as an intermediate phenotype. Experiment Procedure: The yeast RAD9 gene is one putative homolog of the human breast tumor suppressor gene BRCA1. We conducted a genome-wide screen to identify genes that interact with a rad9 mutation to confer enhanced sensitivity to DNA damage. The yeast screen identified 25 interactions, from which we selected 10 candidate gene-gene interactions to be tested in human breast epithelial cells. To determine if the gene-gene interactions discovered in yeast are conserved in human mammary epithelial cells, we built stable human mammary epithelial cells (MCF10A) capable of single- or double-gene knockdown via lentivirus-mediated RNA interference. Cells confirmed for intended gene knockdown were subject to a clonogenic assay to determine if increased sensitivity to DNA damage can be observed in double gene vs. single gene knockdowns. Results: Among the 10 pairs of interactions tested in MCF10A cells, two genes, HLTF and MGMT, were found to interact with BRCA1 to confer enhanced DNA damage sensitivity, based on a clonogenic assay. Cells with combined knockdown of BRCA1 and HLTF show increased sensitivity to a chronic low dose of MMS treatment compared with those with only single knockdown of BRCA1 or HLTF. In addition, distinct morphological features were observed in double knockdown cells with MMS treatment. Cells became enlarged and showed a flat, senescence-like morphology. Even a low concentration of 0.001% MMS induced a strong and irreversible growth arrest in cells with combined knockdown of BRCA1 and HLTF, but not in the single knockdown cells. Conclusion: Our results suggest that it is feasible to use a cross-species (yeast-to-human) comparative systems genetics strategy to identify gene-gene and pathway-pathway interactions. Ongoing studies are aimed at determining whether any of the observed gene-gene interactions contribute to breast cancer penetrance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 229. doi:10.1158/1538-7445.AM2011-229

DNA Damage Discrimination at Stalled Replication Forks by the Rad5 Homologs HLTF and SHPRH

In this issue of Molecular Cell, Lin et al. (2011) describe how HLTF and SHPRH, the human homologs of yeast Rad5, can discriminate between MMS-induced versus UV-induced DNA damage. The results have important implications for the suppression of damage-specific mutagenesis and for the maintenance of genomic stability.

Displaced Strand Regulation of Facxpd Helicase Activity

FacXPD is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) helicase from Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human Superfamily 2 helicase XPD involved in both transcription initiation and nucleotide excision repair, and for the related 5’-3’ helicases FancJ, Rtel and ChlR1 important for maintaining genomic integrity and DNA repair. We developed a single-molecule, high-resolution optical tweezers assay to decipher the mechanism by which a single XPD helicase unwinds dsDNA while translocating in the 5’-3’ direction. This assay monitors the unwinding of an 89-bp DNA hairpin substrate with single base pair resolution. Our substrate design allows us to control the length of a poly-dT ssDNA “translocation” strand (the strand to which XPD binds and along which it translocates), and a “displaced” strand (the strand displaced upon unwinding the duplex), located at the 5’ and 3’ tails of the hairpin, respectively. We found that the displaced strand interacts with XPD and that this interaction controls the helicase activity. When the 3’ tail of the substrate hairpin is substituted for dsDNA, a single XPD molecule displays repetitive “non-processive” bursts of substrate unwinding in which only ∼10-bp of the hairpin is unwound at a time. However, in the presence of a ssDNA displaced strand (of length ranging from 3 to 10 nt), we observe two types of activity: the same 10-bp non-processive mode as above, and also a “processive” mode in which the entire 89-bp hairpin is unwound. These data suggest two different binding modes for XPD resulting in non-processive or processive unwinding which are regulated by its interaction with the displaced strand. We propose a model for how the domains of XPD bind to its DNA substrate in these two modes.

The crystal structure of the ubiquitin-like (UbL) domain of human homologue A of Rad23 (hHR23A) protein

The human homologue of the yeast Rad23 protein, hHR23A, plays dual roles in DNA repair as well as in translocating polyubiquitinated proteins to the proteasome. We determined the three-dimensional structure of its ubiquitin-like (UbL) domain by X-ray crystallography. It has the same overall structure and fold characteristics as ubiquitin and other members of the UbL domain family, with overall root mean square deviations in Cα positions in the range of 1.0-1.3 Å. There are local differences in the α1-β3 loop where hHR23A UbL domain has three more residues constituting a bigger loop. Analysis of the crystal packing revealed a possible dimeric arrangement mediated by the three residues (Leu10, Ile49 and Met75) that are known to be critical for molecular interactions. In contrast to the overall well-defined structure, these three residues are either disordered or have multiple conformations, suggesting that conformation variability is an important property of the binding surface. The electrostatic potentials at the binding surface are conserved among the family, with the hHR23B domain being the most similar to this structure. The intra-molecular complexes formed by the UbL domain of hHR23A with its UbA1 or UbA2 domains was studied by comparative homology modelling, which suggests these two interactions are structurally similar and are mutually exclusive.

DDK Phosphorylates Checkpoint Clamp Component Rad9 and Promotes Its Release from Damaged Chromatin

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombeDbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.

Dissection of the Xeroderma Pigmentosum Group C Protein Function by Site-Directed Mutagenesis

Xeroderma pigmentosum group C (XPC) protein is a sensor of helix-distorting DNA lesions, the function of which is to trigger the global genome repair (GGR) pathway. Previous studies demonstrated that XPC protein operates by detecting the single-stranded character of non-hydrogen-bonded bases opposing lesion sites. This mode of action is supported by structural analyses of the yeast Rad4 homologue that identified critical side chains making close contacts with a pair of extrahelical nucleotides. Here, alanine substitutions of the respective conserved residues (N754, F756, F797, F799) in human XPC were tested for DNA-binding activity, accumulation in tracks and foci of DNA lesions, nuclear protein mobility, and the induction of downstream GGR reactions. This study discloses a dynamic interplay between XPC protein and DNA, whereby the association with one displaced nucleotide in the undamaged strand mediates the initial encounter with lesion sites. The additional flipping-out of an adjacent nucleotide is necessary to hand over the damaged site to the next GGR player. Surprisingly, this mutagenesis analysis also reveals that the rapid intranuclear trafficking of XPC protein depends on constitutive interactions with native DNA, implying that the search for base damage takes place in living cells by a facilitated diffusion process.

Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats

Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5'-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5'-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5'-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5'-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5'-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.

Helicase-like transcription factor confers radiation resistance in cervical cancer through enhancing the DNA damage repair capacity

Helicase-like transcription factor (HLTF) is a member of the SWI/SNF (mating type switching/sucrose non-fermenting) family of ATPases/helicases and also has a RING-finger motif characteristic of ubiquitin ligase proteins. These features have led to suggestions that HLTF functions like yeast Rad5, which promotes replication through DNA lesions via a post-replication repair pathway. However, the function of HLTF in higher eukaryotes is still unknown. Herein, we found the overexpression of HLTF in radiation recurrent human uterine cervical carcinoma tissues when compared to disease free survived patients tissues. In this study, we used RNA interference techniques to investigate the potential function of HLTF in cervical cancer cell line HeLa and found that the cell proliferation was reduced by knockdown (KD) of HLTF. A host-cell reactivation assay showed that the capacity for repair to DNA damage induced by X-ray irradiation was reduced in HLTF KD cells. X-rays also increased apoptosis in HLTF KD cells. These results suggest that HLTF is involved in DNA repair and apoptosis in cancer cells, which might represent a target for gene therapies of human cancer.

Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.

The Yeast E4 Ubiquitin Ligase Ufd2 Interacts with the Ubiquitin-like Domains of Rad23 and Dsk2 via a Novel and Distinct Ubiquitin-like Binding Domain

Proteins containing ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains interact with various binding partners and function as hubs during ubiquitin-mediated protein degradation. A common interaction of the budding yeast UBL-UBA proteins Rad23 and Dsk2 with the E4 ubiquitin ligase Ufd2 has been described in endoplasmic reticulum-associated degradation among other pathways. The UBL domains of Rad23 and Dsk2 play a prominent role in this process by interacting with Ufd2 and different subunits of the 26 S proteasome. Here, we report crystal structures of Ufd2 in complex with the UBL domains of Rad23 and Dsk2. The N-terminal UBL-interacting region of Ufd2 exhibits a unique sequence pattern, which is distinct from any known ubiquitin- or UBL-binding domain identified so far. Residue-specific differences exist in the interactions of these UBL domains with Ufd2, which are coupled to subtle differences in their binding affinities. The molecular details of their differential interactions point to a role for adaptive evolution in shaping these interfaces.

Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant

Rad51 protein promotes homologous recombination in eukaryotes. Recombination activities are activated by Rad51 filament assembly on ssDNA. Previous studies of yeast Rad51 showed that His352 occupies an important position at the filament interface, where it could relay signals between subunits and active sites. To investigate, we characterized yeast Rad51 H352A and H352Y mutants, and solved the structure of H352Y. H352A forms catalytically competent but salt-labile complexes on ssDNA. In contrast, H352Y forms salt-resistant complexes on ssDNA, but is defective in nucleotide exchange, RPA displacement and strand exchange with full-length DNA substrates. The 2.5 Å crystal structure of H352Y reveals a right-handed helical filament in a high-pitch (130 Å) conformation with P61 symmetry. The catalytic core and dimer interface regions of H352Y closely resemble those of DNA-bound Escherichia coli RecA protein. The H352Y mutation stabilizes Phe187 from the adjacent subunit in a position that interferes with the γ-phosphate-binding site of the Walker A motif/P-loop, potentially explaining the limited catalysis observed. Comparison of Rad51 H352Y, RecA–DNA and related structures reveals that the presence of bound DNA correlates with the isomerization of a conserved cis peptide near Walker B to the trans configuration, which appears to prime the catalytic glutamate residue for ATP hydrolysis.

Insights on Rad51 Function from the Structure and Properties of a Filament Interface Mutant

Rad51 protein promotes homologous recombination in eukaryotes. Recombination activities are activated by Rad51 filament assembly on single‐stranded DNA. Previous studies of yeast Rad51 showed that His352 occupies an important position at the filament interface, where it could relay signals between subunits and active sites. Here we report the 2.5A X‐ray crystallographic structure of the H352Y mutant of yeast Rad51. Biochemical data indicate that H352Y is trapped in a high‐affinity ssDNA‐binding conformation with limited catalytic turnover. The structure of H352Y reveals a right‐handed helical filament in a high‐pitch (130Å) conformation with P61 symmetry. The H352Y structure closely resembles DNA‐bound structures of E. coli RecA protein. Tyrosine substitution at position 352 locks Phe187 from the adjacent subunit into a position that interferes with the gamma‐phosphate binding site of Walker A, potentially explaining the limited catalysis observed in H352Y. Comparison of Rad51 H352Y and other RecA/Rad51 structures reveals a correlation between DNA bound/unbound status and the trans/cis configuration of a conserved peptide bond near Walker B.Funded by NIH grant number P01 CA098993.

The Fission Yeast Rad32(Mre11)–Rad50–Nbs1 Complex Acts Both Upstream and Downstream of Checkpoint Signaling in the S-Phase DNA Damage Checkpoint

The Mre11-Rad50-Nbs1 (MRN) heterotrimer plays various and complex roles in DNA damage repair and checkpoint signaling. Its role in activating Ataxia-Telangiectasia Mutated (ATM), the central checkpoint kinase in the metazoan double-strand break response, has been well studied. However, its function in the checkpoint independent of ATM activation, as well as functions that are completely checkpoint independent, are less well understood. In fission yeast, DNA damage checkpoint signaling requires Rad3, the homolog of the ATR (ATM and Rad3-related) kinase, not Tel1, the ATM homolog, allowing us to dissect MRN's ATM-independent S-phase DNA damage checkpoint roles from its role in ATM activation. We find that MRN is involved in Rad3 (ATR)-dependent checkpoint signaling in S phase, but not G2, suggesting that MRN is involved in ATR activation through its role in replication fork metabolism. In addition, we define a role for MRN in the S-phase DNA damage checkpoint-dependent slowing of replication that is independent of its role in checkpoint signaling. Genetic interactions between MRN and Rhp51, the fission yeast Rad51 homolog, lead us to suggest that MRN participates in checkpoint-dependent replication slowing through negative regulation of recombination.

Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis

Previous studies have shown that ubiquitination plays important roles in plant abiotic stress responses. In the present study, the ubiquitin-conjugating enzyme gene GmUBC2, a homologue of yeast RAD6, was cloned from soybean and functionally characterized. GmUBC2 was expressed in all tissues in soybean and was up-regulated by drought and salt stress. Arabidopsis plants overexpressing GmUBC2 were more tolerant to salinity and drought stresses compared with the control plants. Through expression analyses of putative downstream genes in the transgenic plants, we found that the expression levels of two ion antiporter genes AtNHX1 and AtCLCa, a key gene involved in the biosynthesis of proline, AtP5CS, and the copper chaperone for superoxide dismutase gene AtCCS, were all increased significantly in the transgenic plants. These results suggest that GmUBC2 is involved in the regulation of ion homeostasis, osmolyte synthesis, and oxidative stress responses. Our results also suggest that modulation of the ubiquitination pathway could be an effective means of improving salt and drought tolerance in plants through genetic engineering.

PCNA Is Required for Initiation of Recombination-Associated DNA Synthesis by DNA Polymerase δ

Genetic recombination ensures proper chromosome segregation during meiosis and is essential for genome stability and tumor suppression. DNA synthesis after Rad51-mediated DNA strand invasion is a crucial step during recombination. PCNA is known as the processivity clamp for DNA polymerases. Here, we report the surprising observation that PCNA is specifically required to initiate recombination-associated DNA synthesis in the extension of the 3' end of the invading strand in a D loop. We show using a reconstituted system of yeast Rad51, Rad54, RPA, PCNA, RFC, and DNA polymerase delta that loading of PCNA by RFC targets DNA polymerase delta to the D loop formed by Rad51 protein, allowing efficient utilization of the invading 3' end and processive DNA synthesis. We conclude that PCNA has a specific role in the initiation of recombination-associated DNA synthesis and that DNA polymerase delta promotes recombination-associated DNA synthesis.

Biochemical characterisation of the SWI/SNF family member HLTF

HLTF is highly similar in domain organisation to yeast Rad5. We identify PTIP and RPA70, both involved in DNA replication and DNA repair, as HLTF-interacting proteins although cells depleted of HLTF did not show defects in cellular responses to DNA damage. In vitro, HLTF has ATPase activity and E3 ubiquitin ligase activity with a range of E2 ubiquitin conjugating enzymes. HLTF expression is severely reduced in a range of cancer cells, and we suggest that the HLTF antibodies generated in this study could be used for cancer diagnostic purposes.

Nuclear expression of dynamin-related protein 1 in lung adenocarcinomas.

Dynamin-related protein 1 (DRP1), an 80 kDa GTPase, is involved in mitochondrial fission and anticancer drug-mediated cytotoxicity, which implicate an association with disease progression of cancer. In this study we investigated the prognostic value of DRP1 in lung adenocarcinomas. Using immunohistochemistry, we measured the expression of DRP1 in 227 patients with lung adenocarcinomas. Expression of DRP1 was confirmed by immunoblotting. The correlation between DRP1 expression and clinicopathological parameters was analyzed by statistical analysis. Difference of survivals between different groups was compared by a log-rank test. The results showed that DRP1 expression was detected in 202 patients with lung adenocarcinomas. Among these, nuclear DRP1 (DRP1(nuc)) was detected in 184 patients. A significant difference was found in cumulative survival between patients with high DRP1(nuc) levels and those with DRP1(cyt) levels (P<0.001). In vitro, hypoxia increased DRP1(nuc) levels and cisplatin resistance. Antibodies specific to DRP1 co-precipitated a human homologue of yeast Rad23 protein A (hHR23A) and silencing of hHR23A decreased the nuclear DRP1 level and cisplatin resistance. In conclusion, DRP1(nuc) is highly expressed in lung adenocarcinomas, and correlates with poor prognosis. Nuclear DRP1 may increase drug resistance during hypoxia, and hHR23A is essential for nuclear transportation of DRP1. Our results suggest that other than the protein level alone, intracellular distribution of the protein is critical for determining the protein function in cells.

Visualizing the Disassembly of S. cerevisiae Rad51 Nucleoprotein Filaments

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into elongated nucleoprotein filaments on DNA. We have used total internal reflection fluorescence microscopy and a DNA curtain assay to investigate the dynamics of individual Saccharomyces cerevisiae Rad51 nucleoprotein filaments. For these experiments the DNA molecules were end-labeled with single fluorescent semiconducting nanocrystals. The assembly and disassembly of the Rad51 nucleoprotein filaments were visualized by tracking the location of the labeled DNA end in real time. Using this approach, we have analyzed yeast Rad51 under a variety of different reaction conditions to assess parameters that impact the stability of the nucleoprotein filament. We show that Rad51 readily dissociates from DNA in the presence of ADP or in the absence of nucleotide cofactor, but that free ATP in solution confers a fivefold increase in the stability of the nucleoprotein filaments. We also probe how protein dissociation is coupled to ATP binding and hydrolysis by examining the effects of ATP concentration, and by the use of the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido) triphosphate and ATPase active-site mutants. Finally, we demonstrate that the Rad51 gain-of-function mutant I345T dissociates from DNA with kinetics nearly identical to that of wild-type Rad51, but assembles 30% more rapidly. Together, these results provide a framework for studying the biochemical behaviors of S. cerevisiae Rad51 nucleoprotein filaments at the single-molecule level.

An Oligomerized 53BP1 Tudor Domain Suffices for Recognition of DNA Double-Strand Breaks

53BP1, the vertebrate ortholog of the budding yeast Rad9 and fission yeast Crb2/Rhp9 checkpoint proteins, is recruited rapidly to sites of DNA double-strand breaks (DSBs). A tandem tudor domain in human 53BP1 that recognizes methylated residues in the histone core is necessary, but not sufficient, for efficient recruitment. By analysis of deletion mutants, we identify here additional elements in 53BP1 that facilitate recognition of DNA DSBs. The first element corresponds to an independently folding oligomerization domain. Replacement of this domain with heterologous tetramerization domains preserves the ability of 53BP1 to recognize DNA DSBs. A second element is only about 15 amino acids long and appears to be a C-terminal extension of the tudor domain, rather than an independently functioning domain. Recruitment of 53BP1 to sites of DNA DSBs is facilitated by histone H2AX phosphorylation and ubiquitination. However, none of the 53BP1 domains/elements important for recruitment are known to bind phosphopeptides or ubiquitin, suggesting that histone phosphorylation and ubiquitination regulate 53BP1 recruitment to sites of DNA DSBs indirectly.

Repression of the floral transition via histone H2B monoubiquitination

The Rad6-Bre1 complex monoubiquitinates histone H2B in target gene chromatin, and plays an important role in positively regulating gene expression in yeast. Here, we show that the Arabidopsis relatives of the yeast Rad6, ubiquitin-conjugating enzyme 1 (UBC1) and UBC2, redundantly mediate histone H2B monoubiquitination, and upregulate the expression of FLOWERING LOCUS C (FLC; a central flowering repressor in Arabidopsis) and FLC relatives, and also redundantly repress flowering, the developmental transition from a vegetative to a reproductive phase that is critical in the plant life cycle. Moreover, we have found that Arabidopsis relatives of the yeast Bre1, including HISTONE MONOUBIQUITINATION 1 (HUB1) and HUB2, also upregulate the expression of FLC and FLC relatives, and that HUB1 genetically interacts with UBC1 and UBC2 to repress the floral transition. These findings are consistent with a model in which HUB1 and HUB2 specifically interact with and direct UBC1 and UBC2 to monoubiquitinate H2B in developmental genes, and thus regulate developmental processes in plants.