1.

Introduction

In the multi-cellular organisms, a single cell gives rise to functionally specialized

cells, through multiple rounds of cell division to form tissues and organs. This
process is regulated by differential expression of genes at the different time.
Multiple modifications to histone complexes and DNA helps in regulating these
processes. Polycomb group (PcG) proteins in plant and animals are involved in
gene regulation by repressive modification of histone proteins. The PcG pathway
is universally conserved in eukaryotes and mediates epigenetic gene repression
through local chromatin modification and chromatin compaction. PcG mediated
protein repression is stable, heritable and dynamic in response to environmental
and developmental signals.
Polycomb was first identified in Drosophila melanogaster and later other proteins
were discovered which interact in the same pathway and forms multi subunit
Polycomb Repressive Complexes (PRCs). Two major types of PRCs were
described in Drosophila i.e., PRC1, and PRC2. PRC1 constitute four subunits
classified as: Pc, Polyhomeotic (Ph), Posterior sex combs (Psc), and the E3
ubiquitin ligase Sex combs extra (Sce, also called dRing1). Similarly, PRC2 also
constitute of four subunits named as: histone methyl transferase Enhancer of zeste
[E(z)], Suppressor of zeste 12 [Su(z)12], Extra sex combs (Esc), and the histone-
binding nucleosome remodeling factor 55 kDa (Nurf55, also called p55). PRC2
is well conserved across the domain of life. Among PRC2s, homologs of E(z) are
well conserved but the requirement of other three homologs may vary across the
species. On the other hand, PRC1 subunits are less conserved across different
model systems. PRCs are involved in regulating various development processes
ranging from phase transitions, stem cell maintenance and differentiation, cell
reprogramming and many other epigenetic modifications.
In Arabidopsis, three distinct PRC2s have been described: the EMF, VRN, and
FIS complexes. The main role of PRC2 complex is to deposit H3K27me3 marks
at the target loci. These chromatin mark is enriched for the canonical histone
variant H3.1 and depleted of the transcription-associated H3.3 as well as active
marks such as H3K4me3, H3K36me3, and acetylation of lysine on histoneH3
(H3Kac). At the same time, H2Aub is deposited by RING-RAWL twin domain
protein of the RING-RAWUL twin Domain protein of PRC1 complex. These
modification leads to chromatin compaction and helps in gene repression of the
corresponding loci. PRC components usually bind at Polycomb Repressive
Elements (PRE) in the gene body or at the promoter. At least 20% of protein-
coding genes are marked byH3K27me3 in Arabidopsis seedlings which includes
developmental genes expressed in different cell types, hormones, and stress-
responsive genes, suggesting a broad spectrum of processes regulated by PRCs.

PRCs are recruited to their target in a selective way to ensure flexible regulation
of targets at different developmental timings. Studies from Drosophila have
revealed that sequences in gene promoter might help in the recruitment of some
transcription factors (For ex. GAGA factor and pho) which in turn can recruit
PRCs. In mammals, no such consensus sequence has been reported but there is
an overlap between PRC1 & 2 binding sites and CpGIslands. Recent data in
plants suggest that several consensus sequences participate in PRC recruitment.
For some genes, specific sequence elements are known that are required to recruit
PRC2 and initiate H2K27me3 deposition. Such genes include FLC, KNU,
LEAFYCOTYLEDON2 (LEC2), SEPELLATA 3 (SEP3) and many others. The
repressed chromatin mark can are also spread from a nucleation centre to nearby
region. In plants, several transcription factors (TFs) that recruit PRC have been
reported. For ex. ASYMMETRIC LEAVES 1 (AS1) and AS2 directly interact
with EMF subunits and recruit them to the promoters of the KNOX genes BP and
KNOTTED-LIKE FROM ARABIDOPSISTHALIANA 2 (KNAT2) to put
H3K27me3 and repress them during leaf development. Similarly H3K27me3,
H2KA121ub and repression of embryo maturation gene require AtBMI1 protein
which is recruited by VPI/ABI3-LIKE1 (VAL1). The flowering repressors
FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M (FLM) interact
with EMF1 to recruit a putative PRC1-like complex and to establish H3K27me3
and FT repression. Similarly, targeting of LIKE HETEROCHROMATIN
PROTEIN 1 (LHP1) to WUSCHEL (WUS) is mediated by AGAMOUS (AG),
targeting to MAGPIE is mediated by SCARECROW (SCR) and targeting to
SEP3 is mediated by SHORT VEGETATIVE PHASE (SVP). In addition,
RETINOBLASTOMA-RELATED 1 (RBR1) binds to FIE and MSI1 and may
serve to link PRC2 to TFs.
Several recent reports have connected recruitment of Polycomb Repressive
Complexes (PRCs) in plants to cis-motifs and cognate sequence-specific DNA
binding. It has beenhas shown that three related TELOMERE REPEAT
BINDING PROTEINs (TRBs) that recognize teloboxes and telobox related cis-
motifs are crucial for the stable maintenance of H3K27me3 and directly interact
with PRC2 components. TRBs are a plant-specific single-myb-histone group of
proteins. Five isoforms of TRBs have been reported i.e, TRB1-5 and mutations
in trb1 and 3 lead to enhancement of the lhp-1 mutant phenotype in Arabidopsis.
It has been reported that TRB1 is able to bind to chromatin through protein-
protein interaction or sequence non–specific interaction with DNA via its H1/H5
like domain. Previous reports suggest that TRB1 is associated with transcription
start sites (TSS) and involved in ribosome biogenesis. Teloboxes are also present
at the TSS of highly expressed genes that are involved in protein translation, cell-
cycle progression and the Calvin cycle. However, These genes are not usually
H3K27me3 marked and require TRBs to sustain high expression levels. A role of
TRBs in gene repression by PRCs suggests that TRBs should have some
specificity in choosing their targets for H3K27me3 dependent gene repression
and H3K27me3 independent gene activation. It has been suggested that the
presence of a second motif that is recognized by TCP family transcription factors
is required to confer high expression to telobox containing TRB target genes.
Today it is unknown whether different motifs exist that participates in TRB-
mediated PRC recruitment.
2. Aim of the Study
The research focus of this project aims to further our understanding of which
factors determine whether a TRB bound telobox leads to gene PRC2 recruitment
and gene repression vs gene activation. Further, the possibility of a regulatory
unit that consists of teloboxes and secondary, unrelated cis-elements that are
bound by transcription factors that physically interact with TRBs or bridging
complex partners will be explored.
3. Objectives of the Study

(i) Identification of complex partners for TRB1-3 through pull-down and

mass spectrometry analysis.

(ii) In vitro DNA-binding studies and in vivo ChIP-seq for DNA-binding

proteins that co-purify with TRB1-3.

(iii) Bioinformatics analysis to identify potential co-targets of TRBs and

associated transcription factors.

(iv) Generating genome-edited lines that remove binding sites of telobox

associated transcription factors.
4. Methodology

Identification of complex partners for TRB1-3 through pull-down and mass

spectrometry analysis.
Generation of transgenic Arabidopsis plant over expressing TRB 1-3:
As a first step to finding out the protein partners of TRB1-3, coding sequences of
TRB 1-3 will be cloned into a binary vector with a C-terminal GFP-epitope tag.
Transgenic Arabidopsis plants, over-expressing these genes will be generated by
floral dip method using Agrobacterium-mediated gene transformation. The
transgenic plants will be further screened on selection media and will be verified
by semi-quantitative RT-PCR and Western blot immuno detection. Selected lines
will be grown to the next generation. Transgenic Arabidopsis plants expressing
EDS1-GFP in the same vector backbone will be used as a negative control.
TRB 1-3 pull down from transgenic plants:
Total protein isolation and nuclear extract isolation will be done from 4-week-old
transgenic Arabidopsis plants. Specific pull down of the protein will be done by
immuno precipitation with anti-GFP antibody. The successful pull down of the
protein will be further verified by immuno blotting with anti-GFP antibody. Pull
down from EDS1-GFP plants will serve as a negative control.
In-solution mass spectrometry analysis for identification of protein partners:
To further elucidate about the protein partners of TRB 1-3, the immune
precipitated products from transgenic plants will be subjected to
massspectrometry analysis. Briefly, the purified protein will be digested
overnight with modified trypsin. The digested products will be separated by
reverse-phase chromatography and directly injected to the mass spectrometer.
The mass spectrometry data will be analysed to identify the potential protein
partners of TRB 1-3 proteins.
In vitro DNA-binding studies and in vivo ChIP-seq for DNA-binding proteins
that co-purified with TRB1-3
In vitro DNA binding studies:
Pull down and mass spectrometry data will help in narrowing down to the
possible protein partners of TRB 1-3. The coding sequence of partner proteins
that are likely DNA binding proteins will be cloned into expression vectors with
suitable epitope tags and will be purified by chromatographic techniques.
Depending on the available data on or several of the following analyses will be
carried out. For DNA-binding proteins with candidate recognition motifs, DNA
binding properties will be confirmed by an Electrophoretic Mobility Shift Assay
(EMSA). In brief, the DNA oligos will be end labeled with radio labeledγ-p32
ATP and purified proteins will be incubated with the labeled probe in absence
and presence of unlabeled competitor DNA. If no information on the preferred
binding site of a candidate transcription factor is available, a SELEX (Systematic
Evolution of Ligands by EXponential enrichment) experiment will be performed.
Briefly, a pool of random oligomers that are flanked by two distinct anchor
regions will be incubated with the candidate proteins. Protein bound DNA will
be purified and amplified using PCR and primers that anneal to the anchor
regions. After three rounds of amplification and purification, a library for high
throughput sequencing (Illumina) will be prepared. A consensus of the enriched
sequences will be calculated and specific binding to the consensus region
confirmed by EMSA.
In vivo ChIP-seq for identification of DNA binding sites of protein partners:
In order to explore the in vivo binding sites of the proteins that showed binding
to DNA in vitro, in vivo ChIP-seq analysis of these proteins will be performed.
The coding sequence of the potential genes will be cloned with a suitable epitope
tag and transgenic Arabidopsis plants will be generated over expressing these
proteins. Further ChIP will be done with the antibody against the epitope tag.
Sequencing of co-precipitated DNA will be done and the sequencing reads will
be aligned to Arabidopsis genome to get information about the potential binding
sites of these proteins.
Bioinformatics analysis to identify potential co-targets of TRBs and
associated transcription factors.

In order to identify the potential co-targets of TRBs and associated transcription

factor a bioinformatics analysis of ChIP-seq data will be carried out. ChIP-seq
data detecting TRB1-3 target sites are already available and will be compared to
the binding data of associated transcription factors that will be generated during
experiment. ChIP-seq reads will be mapped to the Arabidopsis genome and
significantly enriched regions will be detected. Comparison of the peak profiles
of TRB1-3 and the new transgenic plants will be compared will help in
identifying potential co-targets of the complex. Statistical analysis will also be
performed to compare the frequency of potential transcription factor binding sites
(TFBS) for TRB1-3 and the interacting DNA-binding proteins. Furthermore,
target genes will be analysed for enrichment of specific chromatin modifications,
expression profiles and gene function (using gene ontology terms). We are
particularly interested in transcription factors and TFBS that bind to H3K27me3
target genes in combination with TRB 1-3.

Generating genome-edited lines that remove binding sites of telobox

associated transcription factors.

Once potential binding sites of TRB 1-3associated transcription factor and

co-target genes are identified, gene-edited lines of Arabidopsis will be generated
that carry mutations in the associated TFBS by using the CRISPR-Cas9 genome
editing method. I will be most interested in co-bound target genes that are also
controlled by the plant PcG pathway. These mutant plant lines will be used to
validate the role of associated TFBS in telobox associated transcription factor
recruitment. Briefly, the guide RNAs against the binding site along with Cas9
will be cloned into a binary vector that expressed CAS9 early in embryo
development. Arabidopsis transgenic lines will be generated using these
constructs and T1 transgenic plants will be screened for editing events at the
target regions by a PCR based approach. Plant lines with editing events will be
propagated into the next generation for the selection of stably inherited
homozygous mutations and the absence of the CAS9 transgene.

Selected mutated lines will be further investigated by comparing target gene

expression to that of wild-type plants and by comparing the H3K27me3 profiles
by ChIP-PCR. Co-dependence of TRB 1-3 and interaction partners for binding of
target sites can be tested using available antibodies against TRB1 and TRB3 in
ChIP experiment.

From this research, we are expecting to deepen our understanding of how PRCs
are recruited to their target sites in Arabidopsis. Identification of sequence motifs
and protein partners of TRB 1-3 will help to explain how TRBs are involved in
gene repression vs. gene activation. It will further help us to understand the
mechanism of gene repression by PRC complex in plants. Together these results
will help in a more clear view of gene repression in plants by PRCs.