1. My Undergraduate Research Summary
My undergraduate research project in the Marzluff
lab explored the regulation of histone proteins, which
form the structural basis of chromatin—the packaging
material of eukaryotic DNA.1
DNA replication and
histone protein synthesis are synchronized and occur
together during S phase of the cell cycle—so that
chromatin is available for newly synthesized DNA
molecules. The synchronization of histone mRNA
synthesis and the cell cycle is mediated by a unique,
non-polyadenylated, 3 ’ end histone mRNA processing
signal. In contrast to all other known eukaryotic
mRNAs, histone mRNAs have a stem-loop processing
signal and a histone downstream element (HDE),
which are evolutionarily conserved across all
metazoans. As a result of the 3’ stem-loop structure
and HDE, histone mRNAs are processed and degraded
in a cell cycle dependent manner that is different from
the processing and degradation of polyadenylated
mRNAs.2
My research project aims to answer two
fundamental questions: (1) why have histone genes
evolved a unique processing signal, different from all
other known eukaryotic mRNAs? and (2) why is the
stem-loop structure so highly conserved in metazoans?
In Drosophila melanogaster the histone genes are
clustered at a single locus in the genome, making it
relatively easy system to genetically manipulate. The D.
melanogaster histone genes are arranged in a series of
about 100 copies of a tandemly arranged 5kb repeat
containing a single copy of each of the five canonical
histone genes (H1, H2A, H2B, H3, and H4)—though
just 12 copies of the repeat have been shown to rescue
from lethality and confer fertility. A histone
replacement platform has been developed that can be
use to produce mutant D. melanogaster with any
desired histone genotype. This platform works by
introducing twelve repeat (12X) arrays of transgenes
with specifically engineered histone genotypes into
fly lines in which the entire histone cluster has been
deleted (Figure 1).2,3
The Marzluff lab has previously designed a DNA
sequence encoding the five canonical histone genes of
D. melanogaster, with the addition of eighteen
strategically placed restriction enzyme sites, known as
the Designer Histone Locus (DHL). These restriction
sites were placed before each start codon, after each
stop codon, after each HDE, and on the far ends of the
sequence—allowing for easy manipulation of the
sequence (Figure 1). Prior to the start of my project, the
DHL had been altered using molecular cloning
techniques to contain a strong polyadenylation signal in
place of the endogenous H2A stem-loop sequence and
HDE (H2ApA). Thus my project has so far focused on
synthesizing a 12X array of the H2ApA construct.
Using molecular cloning techniques (such as,
restriction digests, gel electrophoresis, DNA ligations,
genetic transformations, cell culturing, and plasmid
preps) I first arrayed the H2ApA first from 1Xà2X,
then from 2Xà4X, 4Xà8X, and finally 8Xà12X.
Successful arraying of my 12X construct was
confirmed with gel electrophoresis (Figure 2). My 12X
H2ApA construct will be introduced into D.
melanogaster, along with a phenotypic marker used to
indicate transgene insertion. Once screened, these flies
will provide a system for studying the consequences of
a polyadenylated canonical histone gene. Furthermore,
transgenic flies will be crossed with a fly line in which
endogenous histone cluster has been deleted. If my
engineered histone construct rescues from lethality,
then a line of flies homozygous for deletion of the
endogenous histone cluster and containing the
transgenic 12X array will be generated for further
study.
Figure 1. Left column. Map of the Designer Histone Locus (DHL)
containing the five canonical histone genes of D. melanogaster,
along with the addition of eighteen restriction enzyme sites. These
restriction sites were inserted before each the start codon (green),
after each stop codon (red), and after each HDE (blue). A twelve
repeat array of the DHL, containing an H2A polyadenylation
signal, was successfully arrayed.
References: (1) Annunziato, A. Nat. Edu. 2008, 1, 26. (2) Marzluff, W.; Wagner, E.; Duronio, R. Nat. Rev. Genet. 2008, 9, 843. (3) McKay, D.;
Klusza, S.; Penke, T.; Meers, M.; Curry, K.; McDaniel, S.; Malek, P.; Cooper, S.; Tatomer, D.; Lieb, J.; Strahl, B.; Duronio, R.; Matera, G. Dev.
Cell. 2015, 32, 373.