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Colourful spot, stripe and ring patterns decorate the corolla of many flowering plants and fulfil important biotic and abiotic functions. These petal patterns are created by spatial differences in pigmentation, cell shape and texture of the adaxial petal epidermis. The mechanisms controlling formation and evolution of these patterns, and their exact role in plant-animal communication are not well understood. My PhD adds to current understanding of petal patterns by investigating the development, evolution, and function of the bullseye pigmentation pattern on Hibiscus trionum petals. The bi-coloured bullseye pattern of H.trionum is created from contrast in cell shape, cuticle texture, and pigmentation between the bottom and top of the petal. The bottom petal appears shiny and purple because cells in this region are elongated with a striated cuticle, and are pigmented with anthocyanins. A minimal regulatory network restricting anthocyanin pigment production to the bottom of Hibiscus trionum petal was identified. The network relies on two MYB regulators: HtCREAM1 which represses anthocyanin production in the top of the petal; HtBERRY1 which promotes anthocyanin biosynthesis in the bottom of the petal. This minimal network is a starting point to understand the molecular mechanisms both creating, and creating diversity, in petal patterns. Natural variation in the bullseye pattern in close relatives of H.trionum from Australia and New Zealand is in part due to four independent restrictions of anthocyanin pigmentation in the petal bottom. Preliminary results suggest restriction of pigmentation in H.richardsonii, sister-species to H.trionum, results from mutation in the regulatory region and the coding sequence of the BERRY1 homolog of H. richardsonii. Natural variation in the bullseye pigmentation pattern could reflect a function in plant-pollinator communication. Buff-tailed bumblebees were found to discriminate between, and prefer, artificial flowers with H.trionum-like bullseye patterns to H.richardsonii-like bullseye patterns.

Tissue-resident macrophages (MΦ) such as microglia, Kupffer and Langerhans cells derive from Myb-independent yolk sac (YS) progenitors generated before the emergence of hematopoietic stem cells (HSCs). Myb-independent YS-derived resident MΦ self-renew locally, independently of circulating adult monocytes and HSCs. In contrast, adult blood monocytes as well as infiltrating, gut and dermal MΦ derive from Myb-dependent HSCs and are less proliferative. These findings are derived from the mouse, using gene knock-outs and lineage tracing, but their applicability to human development has not been formally demonstrated. Here I use a human pluripotent stem cell (hPSC) differentiation model of hematopoiesis, capable of monocyte/MΦ production over prolonged periods of time, as a tool to investigate human mononuclear phagocyte ontogeny. Using a transcriptomic approach I showed that hiPSC-derived monocytes/MΦ (iPS-Mo/MΦ) produced early in differentiation (first weeks) are more proliferative and less immunologically mature than iPS-Mo/MΦ produced at a later time point. I therefore hypothesised either that iPS-Mo/MΦ only become fully mature after several weeks of differentiation or that there are two developmentally distinct waves of MΦ produced over time. By comparing the transcription profile of iPS-Mo/MΦs to that of primary adult blood monocytes and fetal microglia I then showed that early and late iPS-Mo/MΦs were transcriptionally closer to fetal microglia than to adult blood monocytes. To further investigate if iPS-Mo/MΦs are indeed of the same developmental origin as MYB-independent MΦ such as microglia, I used a CRISPR-Cas9 knock-out strategy to show for the first time, that human iPS-Mo/MΦs develop in a MYB-independent, RUNX1 and SPI1 (PU.1)-dependent fashion. This result makes human iPS-Mo/MΦs developmentally related to, and a good model for, MYB-independent tissue-resident \Macros such as alveolar and kidney MΦs, microglia, Kupffer and Langerhans cells. Interestingly, while MYB was not required for the generation of iPS-Mo/MΦs, its knock-out resulted in an increase in iPS-Mo/MΦ production. To investigate this increase I developed two methods for quantifying the differentiation bottleneck occurring during hiPSC differentiation to iPS-Mo/MΦs. Those techniques highlighted a potential increase in progenitor cell generation in MYB KO cells and thus lay foundation to improve our technical understanding of EB differentiation and will enable enhanced manipulation of the EB model.

Flowering dogwood (Cornus florida L.) and Asian dogwood (C. kousa F. Buerger ex Hance) are popular understory and deciduous ornamental trees that are mostly found throughout the temperate regions of North America, Europe, and Asia. They are valued for their showy snow-white, pink, and red bracts, red fall foliage, and colorful fruit. Ornamental dogwoods are essential horticultural crops for the economy of Tennessee, Oregon, North Carolina, and Ohio. Tennessee-based growers led the trade and accounted for 24% of US sales of flowering dogwood in 2019, whereas total dogwood sales exceeded USD31 million annually. Although more than 150 cultivars are commercially available for both species, consumer demands for specific traits of interest, such as dark red bract or foliage display, resistance to pathogens, variegated foliage, and dwarf or weeping habit, have not been satisfied. However, time-demanding dogwood cultivar development typically depends on repeated selection for desirable traits. Hence, the following challenges must be overcome: long juvenile period (3 to 5 years), self-incompatibility, inbreeding depression, and high susceptibility of flowering dogwood to two fungal foliar pathogens, dogwood anthracnose and powdery mildew. Therefore, this work was initiated to study the molecular mechanisms underlying anthocyanin and proanthocyanidin pigment biosynthesis; to enhance bract and foliage's desirable red and pink colors by using the RNA-Seq approach to enable the development of functional markers or the application of marker-assisted selection. Fully developed bracts of three cultivars of C. florida and two cultivars of C. kousa were sampled when color was maximally visible and subjected to RNA sequencing. Candidate genes CfMYB2, CfMYB3, and CkMYB2, were found to repress anthocyanin and proanthocyanidin synthesis in flowering and Asian dogwood, whereas CfMYB1 80 and CkMYB1 increased it. Two quantitative trait loci, QTLPM-7 and QTLPM-8, associated with powdery mildew resistance were identified for two consecutive years in C. florida pseudo-F2 population. Finally, recessively inherited Mildew resistance locus o (Mlo) genes, which prevent effective host cell penetration by powdery mildew, were investigated in a time-tuned transcriptome study including four C. florida cultivars of varying resistance to powdery mildew.

The fetal to adult hemoglobin switch is a developmental process by which fetal hemoglobin becomes silenced after birth and replaced by adult hemoglobin. Diseases caused by defective or missing adult hemoglobin, such as Sickle Cell Disease or β-Thalassemia, can be ameliorated by reactivating fetal hemoglobin. We discovered that knockdown or knockout of β-globin, a subunit of adult hemoglobin, led to robust upregulation of γ-globin, a subunit of fetal hemoglobin. This phenomenon suggested that red blood cells have an inherent ability to upregulate fetal hemoglobin in the event that adult hemoglobin is lacking.We developed multiple gene-editing tools in an immortalized erythroid cell model to investigate the molecular mechanisms behind the increase in fetal hemoglobin. Time-course transcriptomics identified ATF4, a transcription factor, as a causal regulator of this response. Further analysis also converged upon downregulation of MYB and BCL11A, known repressors of γ-globin, described in detail in chapter 2. Further work in chapter 3 explores other possible fetal hemoglobin regulators as discovered by CRISPRi arrayed mediated knockdown experiments. This work furthers our understanding of fundamental mechanisms of gene regulation and how cellular and molecular events influence red blood cell differentiation.

Salivary gland adenoid cystic carcinoma (ACC) is an aggressive tumor with a tendency to infiltrate surrounding nerves and metastasize to distant sites. The standard treatment often fails to control local tumor recurrence and distant metastases and no approved targeted therapeutic options exist for these tumors. The goal of our studies was to reveal the molecular mechanisms driving ACC tumor development and novel drug targets to improve patient morbidity and mortality. We first analyzed clinical and RNA-sequencing (RNA-seq) data for 68 formalin-fixed paraffin-embedded (FFPE) ACC tumor samples and described previously unappreciated molecular heterogeneity that predicts patient outcome. The poor outcome subgroup had a gene expression signature that resembled embryonic stem cells, suggesting these patients had high-grade dedifferentated tumors. We also utilized these RNA-seq data to definitively show that the MYB and MYBL1 genes are the oncogenic drivers in the vast majority of ACC tumors. ACC tumors that expressed Myb oncogenes were distinct from those that did not, indicating that Myb driven tumorigenesis in ACC tumors results in a unique gene expression pattern. From these analyses we identified and validated the first two high-confidence Myb regulated genes in ACC tumors, the first step in unraveling how Myb driven gene expression changes drive oncogenesis. The MYB gene must be truncated to unleash its oncogenic potential, in ACC tumors this typically occurs at the C-terminus via chromosomal translocations. However we only observed gene truncation in half of the MYB expressing ACC tumors, raising a new question: how is the oncogenic potential of MYB unleashed in those tumors that appear to express the full-length gene? We found that nearly all of the MYB expressing ACC tumors had an activated alternative MYB gene promoter, which produces N-terminally truncated Myb proteins. Thus, alternative promoter use may unleash the oncogenic potential of the Myb protein in those ACC tumors that appeared to express the non-oncogenic full-length protein. Further investigation revealed significant differences in the gene expression signature elicited by N-terminally truncated Myb isoforms and full-length Myb isoforms. Specifically, N-terminally truncated Myb isoforms uniquely activated a pro-tumorigenic neural migration signaling pathway. A pathway linked to increased perineural invasion, an ACC tumor hallmark associated with poor prognoses. Indeed, we found stratification of ACC tumors by expression of these genes identified a significantly poor outcome subgroup. The clinical heterogeneity that makes ACC tumors so difficult to treat may be predicted by molecular characterization, thus identifying high-risk patients who are candidates for more aggressive care or personalized therapies. A previously unidentified truncated Myb isoform stimulates perineural invasion, a significant roadblock to curative treatment. These studies have many implications for future studies of the mechanisms of Myb driven tumorigenesis, development of more effective targeted therapeutics, and ultimately patient treatment.

Glyceollin phytoalexins are the pathogen-elicited defense metabolites that belong to the isoflavonoid family of molecules of soybean (Glycine max L. Merr). Phytophthora root and stem rot of soybean caused by Phytophthora sojae is a destructive disease throughout the soybean-growing regions worldwide causing devastating economic damages (globally $1-2 billion and over $250 million in the USA) every year. Engineering soybean plants that produce higher levels of glyceollins could confer resistance against this pathogen. Glyceollins also show anticancer and neuroprotective activities in mammals, therefore they are important for agriculture and medical research. Firstly, we were interested in understanding how biotic and chemical elicitors regulate glyceollin biosynthesis differently. We found that combining the biotic elicitor WGE (wall glucan elicitor) extracted for P. sojae and the chemical elicitor silver nitrate had an additive effect on the elicitation of glyceollins I. They elicited glyceollins by separate biosynthetic mechanisms. We treated soybean seedlings with a panel of abiotic stress conditions and found that acidic medium elicited the highest amount of glyceollins in root tissues of 1700µg gt-1 (gram tissue, Fresh Weight). Then we focused on the identification of transcription factors (TFs) that regulate the biosynthesis of glyceollins in response to abiotic and biotic stress. Based on a comparative transcriptomics approach using RNA-seq, we chose to functionally characterize the NAC (NAM/ATAF1/CUC2) family TF GmNAC42-1, a homolog of the camalexin regulator ANAC042 from Arabidopsis and an R2R3-MYB (myeloblastosis)-type TF GmMYB29A2, the homolog of the stilbene regulator VvMYB14 from grapevine. Overexpressing and RNAi silencing of GmNAC42-1 and GmMYB29A2 in WGE-elicited hairy roots resulted in significantly increased and decreased accumulation of glyceollin metabolites and biosynthesis gene transcripts such as glycinol 4-dimethylallyl transferase (G4DT) and isoflavone synthase2 (IFS2). The GmNAC42–1 and GmMYB29A2 proteins directly bound the promoters of glyceollin biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system and localized to the nucleus when expressed with an N-terminal green fluorescent protein (GFP) tag. EMSA (Electrophoretic Mobility Shift Assay) confirmed that MYB29A2 protein binds glyceollin gene promoters. Overall, our findings indicate that GmNAC42–1 and GmMYB29A2 are essential components of a glyceollin gene regulatory network (GRN) and could be used for the large-scale, economic bioproduction of glyceollins for therapeutic use and to enhance resistance against P. sojae.

Abstract: Fiber harvested from flax phloem tissue is a renewable resource with promising uses in eco- friendly composites. Most molecular and cellular research to date has focused on later stages of fiber differentiation including the development of the fiber cell wall. On the other hand, the molecular mechanisms that govern specification of fibers are largely unknown. All phloem fibers in flax are formed during primary growth. Therefore transcription factors enriched in the shoot apices are likely to govern fiber identity, and therefore fiber yield. In this study, I used RNA-Seq to compare the gene expression in the apical region (AR) of the shoot apex which contained the apical-most 0.5mm of the stem and basal region (BR), which contained the entire stem except for the apical-most 1 cm. AR included the SAM and its immediate derivatives whereas BR represented all stem and vascular tissues at later stages of differentiation. The RNA-Seq study identified 349 putative transcription factors that are preferentially expressed in the AR including 18 MYBs and nine NACs. MYBs and NACs have been revealed to be required for the vascular cell identity in other species. A total of 240 putative MYBs and 182 predicted NAC domain genes were identified within the whole-genome sequence of flax. Phylogenetic analysis of the flax NAC gene family revealed that two distinct subfamilies were largely expanded. Flax had a higher proportion of R2R3-MYB than most of other sequenced plant species. Analysis of the expression data in public database indicated that the majority of LusMYBs and LusNACs were expressed in wide range of tissues with low expression level while a few others were particularly abundant in some specific tissues. Transcript expression profiling of the LusNACs in the VNS subfamily in 12 different flax tissues suggested that LusNAC28 and LusNAC125 were highly expressed in developing fibers. A previously uncharacterized Arabidopsis gene, At3g05980, encodes a predicted protein of 245 amino acids (27.6 kDa). This protein does not contain any annotated domains, and its predicted secondary structure consists mostly of disordered coils. It has one closely-related paralog in Arabidopsis, At5g19340. Homologs of At3g05980 are found in all eudicots examined, but not in any other taxa. There are four highly conserved amino acid motifs within the protein. Using qRT- PCR and GUS reporter assays, I found that transcripts of At3g05980 were highly expressed in immature embryos and the micropylar endosperm, as well as petals, and apices of shoots and roots, and atrichoblasts. Transcripts were highly induced by cold treatment, but not by other stress or hormone treatments. These results were consistent with expression patterns previously reported in public databases. I produced loss-of-function (LOF) mutants of this gene, using CRISPR/Cas9- mediated gene editing, as well as overexpression (OX) lines using the 35S-CaMV promoter. LOF lines were morphologically indistinguishable from wild-type, but OX lines had minor defects, including cotyledon epinasty, and slight shortening of both plant height and silique length. Neither LOF nor OX differed from WT in tolerance to freezing. In the absence of cold-treatment, LOF mutants had increased transcript abundance of the stress- and cold-responsive gene RD29, compared to WT, but expression patterns of five other cold-responsive genes were largely unchanged in LOF, compared to wild-type, both before and during cold treatment. Translational fusions of At3g05980 with fluorescent proteins were localized to peroxisomes. However, assays of peroxisomal function, including dark growth of seedlings, and sensitivity to 2, 4-DB and IBA, were similar between LOF, OX, and WT. Furthermore, fatty acid profiling of seeds did not show any difference between the genotypes. Thus, At3g05980 encodes a eudicot-specific, peroxisomaly localized protein with transcripts that are cold-inducible, and enriched in specific tissues (particularly rapidly growing tissues), but this gene does not appear to be required for normal morphology, peroxisomal function, or cold tolerance responses. The immediate future task will be to examine phenotypes in double mutants of both At3g05980 and it paralog At5g19340.

MYB transcription factors (TFs) serve many important regulatory roles in plants, making them an important topic of study. However, like many large gene families, there are many inherent difficulties in understanding both the individual roles, and evolutionary history of MYB TFs. Individually, it may be difficult to isolate the functions of a given MYB TF, as there are numerous similar TFs serving related or overlapping functions. As a group, the similarities between members of a gene family can make it difficult to disentangle the forces causing their propagation and maintenance. There are many essential pathways governed by TFs that have evolved via massive expansions, such that a high degree of similarity and redundancy occurs within the gene family. This thesis is concerned with confronting this complex issue, attempting to learn as much as possible about the history and patterns found within the MYB TF family. The objectives of this research are to examine the results of screens involving MYB TF binding partners, and to apply new tools and methods to MYB genes to learn about their evolutionary history. Based on the meta-analysis of biological screens and the use of phylogenetic and recombination detection techniques, this research proposes mechanisms to explain patterns observed and improve the predictive power of computational approaches.

AbstractThe Development and Evolution of Floral Symmetry in the Zingiberales and Interactive Tools for Teaching Evolution (ArborEd)byRiva Anne BruennDoctor of Philosophy in Plant BiologyUniversity of California, BerkeleyProfessor Chelsea D. Specht, ChairFloral symmetry is a key innovation in the evolution of flowering plants. Zygomorphy, or single-planed symmetry, is associated with the diversification of many flowering plant lineages. The model system for floral symmetry is the snapdragon (Antirrhinum majus). In A. majus flowers, a set of TCP and MYB-related transcription factors form a core gene regulatory network necessary for zygomorphy. The genes involved in this network have been implicated in several independent transitions to zygomorphy from actinomorphy (many-planed symmetry). Although the TCP components of the symmetry network have been investigated across flowering plants, MYB-related transcription factors remain largely unstudied outside of the Asterid group containing A. majus and close relatives. Here we investigate the evolution of MYB-related genes DIVARICATA-like (DIV-like), RADIALIS-like (RAD-like), and DIVARICATA and RADIALIS INTERACTING FACTOR-like (DRIF-like) across flowering plants, and their expression patterns in the developing flowers of two zygomorphic species of the monocot order Zingiberales. We found that RAD-like and DIV-like are sister MYB-related genes which diverged before the diversification of flowering plants. Each gene contains one MYB-like domain that has been closely conserved throughout flowering plant evolution. Furthermore, we identified candidate homologs to A. majus RAD and DIV in several monocot taxa, with at least three copies of each in the Zingiberales. In the Zingiberales, RAD-like and DIV-like genes are expressed in Costus spicatus (Costaceae) and Musa basjoo (Musaceae) in patterns consistent with roles in floral symmetry. Using Reverse Transcription PCR and in situ hybridization we recovered asymmetric expression patterns for some RAD-like genes across the dorsal/ventral plane of developing flowers, and universal expression of DIV-like genes, consistent with the model known from Antirrhinum majus. We identified DRIF-like genes across flowering plants, recovering a previously undescribed duplication in eudicot DRIF Group 1 genes. Furthermore, we recovered candidate DRIF-like genes in Musa basjoo (Musaceae: Zingiberales) with expression patterns similar to those described in A. majus DRIF1 and DRIF2. Finally, we developed a tutorial for high school and college students to investigate a coevolutionary hypothesis in sharpshooters and their bacterial endosymbionts. This tool will help students understand how comparative evolutionary research is performed, and give them hands-on experience performing common analyses.

The unveiling of small RNA (sRNA)-mediated gene regulatory pathways has profoundly shaped our understanding of the complexity of gene regulation. In eukaryotes, sRNAs have been found to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat viruses and mobile genetic elements. To gain insight into the roles of small RNAs in apple and peach, we conducted sRNA-seq, computational analysis and molecular experiments to genome-widely characterize their microRNAs (miRNAs) and trans-acting siRNA (tasiRNA) pathways. We identified totally 75 miRNAs or families, including 23 conserved, 10 less-conserved and 42 apple-specific ones, and 118 miRNA target genes in apple. Two classical trans-acting siRNA (tasiRNA) pathways, miR390-TAS3 and miR828-TAS4, were characterized with similar but unique tasiRNA biogenesis profiles and target specificities. Importantly, miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. In contrast to the location of the miR159 target site in a sequence-divergent region, the target sites of miR828 and miR858 are located in the region encoding the conserved R3 repeat domain of MYB proteins. 10 out of the 19 miR828-targeted MYBs undergo the biogenesis of various phased siRNA (phasiRNA), which potentially regulate diverse genes outside the MYB family. In peach, totally 94 miRNAs or families and 80 target genes were identified. Similar pathways of tasiRNA (miR828-TAS4 and miR390-TAS3) or phasiRNA (miR828-MYB-siRNA) processing were also characterized in peach. Taking advantage of reverse computation and public available deep-sequencing data, we demonstrated that the miRNA-TAS-PPR-siRNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to instigate phasiRNA production from PPR �genes, which are triggered by different 22-nt miRNAs, including miR7122, miR1509, and fve-PPRtri1/2 and through distinct mechanistic strategies, like miRNA direct-targeting or indirect-targeting through TAS-like genes, one-hit or two-hit, or even two layers of tasiRNA-TAS interactions. We found that the MIRNA genes of these miRNA triggers show great identity with the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Combined results from phylogenetic analyses and conservation extent profiling revealed that the super-miR7122 was potentially evolved from another miRNA superfamily (super-miR4376), which probably originated from the miR390. Additionally, the miR482/2118-NB-LRR-siRNA pathway was found to be conserved, but evolved with distinct features, in apple and peach. Taken together, widespread and complex miRNA and tasiRNA regulatory networks have been adapted in apple and peach. They add another crucial layer of regulation on gene activity and stability, and must exert essential functions in all aspects of plant life.

In the model cruciferous plant, Arabidopsis thaliana, tryptophan (Trp) is a focal point for growth and defense as it is used for the production of secondary metabolites including the growth hormone indole-3-acetic acid (IAA, or auxin), and two classes of defense compounds: indole glucosinolates (IGs) and camalexin. Trp metabolism in plants is of general importance to agriculture because animals (including humans) cannot synthesize Trp and must obtain it from their diet. Questions remain about the synthesis and regulation of Trp and how it relates to secondary metabolism in Arabidopsis. In this thesis it is shown that IGs are a sink for Trp metabolism because auxotrophic mutants deficient in Trp production are suppressed in combination with the IG-deficient cyp79B2 cyp79B3 mutant and enhanced in combination with IG overproducing mutant, atr1D. Because Trp auxotrophic mutants were found to produce IGs, the four predicted Arabidopsis Trp Synthase Beta genes (TSB1, TSB2, TSB3 and TSBt2) were examined for their role in Trp primary and secondary metabolism. It was determined that members of this gene family, while being redundant for enzyme activity, may have unique functions in channeling Trp to different secondary endpoints. tsb1 tsb2 plants display a healthier phenotype and produce lower IG levels than the single tsb1 mutants, in contrast to tsb1 tsbt2 plants, which have elevated IG production and an enhanced auxotrophic phenotype. tsb2 tsbt2 plants are indiscernible from WT. Gene expression in Trp biosynthetic pathway steps, IG biosynthesis genes, and regulatory TFs is dysregulated in these mutants. In a second part of this thesis, transcriptional regulation of IG synthesis was examined with respect to tissue specificity and stress. In collaboration with Judith Bender's laboratory at Brown University, the function of a subfamily of three Myb transcription factors that have been implicated in regulating IG biosynthesis genes was studied. Using combinations of Myb knockout mutants and GUS reporter plants, tissue specific roles for MYB34 and MYB51 in root and shoot tissues, respectively, were found. In addition, roles were discovered for MYB34 in mediating anti-herbivory signals, and for both MYB51 and MYB122 in regulating defense against microbial pathogens.

The maize flavonoid biosynthetic pathway has two distinct branches regulated by two very similar MYB domain transcription factors. C1 regulates the accumulation of anthocyanins while P1 regulates the synthesis of phlobaphenes. These two proteins exhibit more than 70% identity in the amino acid sequence of their MYB domain, bind the same DNA sequences in vitro, albeit with different affinity, and share a set of common target genes. One difference between these proteins is that C1 is absolutely dependent on the bHLH cofactor R for activity while P1 is not. We have shown that it is through the interaction with R that C1 achieves specificity. We have also found that the function of R can be dissected into an R-enhanced activity and an R dependent activity. The R-enhanced activity is achieved through the ARE, a cis-DNA element present in all the C1 target genes. I propose that R recruits a DNA binding factor that recognizes and binds the ARE. The R-dependent activity is an effect that R has on the C1 protein that allows it to bind and activate its target promoters. Due to its multidomain structure, R is likely to recruit additional factors to the regulatory complex. We have identified an ENT domain protein, RIF-1C, which specifically interacts with the bHLH region of R, and uncovered evidence that suggests that the bHLH and RIF-1C are involved in chromatin remodeling.