Conservation and
Diversification of Three-Repeat
Myb Transcription Factors in
Plants
Masaki Ito
Presented By Nina Carini and Erika Thiele
Objective
The purpose of this study is to show that
Myb transcription factors containing three
repeat sequences in their DNA-binding
domains have a significant role in
regulating transcription of cyclin B in the
G2/M phases of tobacco.
What is Myb ?
Myb is derived from “myeloblastosis”, which is a name
for a specific type of leukemia.
This gene was first recognized as the v-Myb oncogene
of the avian myeloblastosis virus.
Family of transcription factors containing 2 or 3 repeat
sequences in the DNA-binding domain (Myb domain).
Each repeat sequence is composed of approximately
50 amino acids.
These proteins bind to MSA elements in the promoter
regions of specific genes such as cyclin B and activate
transcription late in the G2 phase.
The domains that contain three repeat sequences have
been shown to be evolutionarily conserved among
plants. This study will focus on proteins that contain
three repeat Myb domains.
Myb Structure
The structure of Myb domains are represented
as a helix-turn helix formation.
This structure contains a domain with three
repeat sequences (in blue).
The Cell Cycle
• S phase: DNA replication
• M phase: Mitosis and Cytokinesis. Mitosis has 4 stages:
prophase, prometaphase, metaphase, and anaphase.
Cytoplasm division occurs telophase.
Arabidopsis Genome
5% of the Arabidopsis genome encodes 1,500
transcription factors.
One of the largest families is the Myb family.
Arabidopsis contains 130 Myb genes and 125
of these genes encode proteins with two repeat
sequences. The proteins of interest contain
Myb domains with three repeat sequences.
Arabidopsis contains 9 proteins that encode
cyclin B.
Cyclin B genes in Arabidopsis
Methods
Five 3R Myb proteins were isolated from
tobacco.
NtmybA1, NtmybA2, and NtmybB were
isolated by yeast one-hybrid screening.
NtmybC1 and NtmybC2 were isolated by
PCR.
Yeast One-Hybrid
Screening
Two yeast strains carrying the HIS3 reporter
gene with a six repeat MSA element and three
repeat MSA element were constructed.
The yeast cells with the six repeat MSA
element were transformed with cDNA
fragments of mRNA that were prepared from
actively dividing tobacco cells.
The yeast cells were cultured to obtain
colonies with the HIS3 reporter gene.
Polymerase Chain
Reaction
PCR amplified the specific sequences of NtmybC1 and
NtmybC2.
In plants, myb factors comprise one of the largest
families of DNA-binding domains.
DNA sequences for NtmybC1 and NtmybC2 have not
been determine.
Amino acid sequences are similar.
Electrophoretic mobility
shift assay
To test if NtmybA1,
NtmybA2, and NtmybB
bind specifically to MSA
elements.
Mutations in MSA
element prevent
binding of proteins.
Binding of proteins to MSA
element
RNA Gel Blot
To determine
accumulation of
NtmybA1,NtmybA2, and
cyclin B late in G2
phase.
NtmybA1, NymybA2,
NtmybB, and cyclin B
were treated with
aphidicolin, a substance
that inhibits the cell
cycle. Upon removal of
the aphidicolin, the
proteins were run on a
gel.
Gene expression late in
G2 phase
Autoregulation: NtmybA1 and
NtmybA2 have their own
promoters. They can bind to
their own promoters and
enhance their activity.
Cyclin B binds to CDK to form a
complex and phosphorylate
NtmybA2.
Future Studies
Comparison of plant 3R Myb proteins and
animal 3R Myb proteins.
To determine which gene sequences NtmybC1
and C2 interact with.
The mechanism for the positive feedback loop
of NtmybA1 and NtmybA2 proteins with cyclin
B.
The mechanism that causes accumulation of
Ntmyb proteins and cyclin B late in the G2
phase.