Automatic Analysis of BrdU Incorporation for Replication Timing Assays

Intern: Albert F. Cervantes

Advisors: York Marahrens Moira Regelson Silvia Diaz-Perez

Overview

• Motivation

• Background

• The Project

• Future Work

Motivation

• Marahrens’ Lab studies X-inactivation– Only one X-chromosome necessary for

survival (e.g. males)– One X-chromosome must be inactivated

• Dosage control: female with two X-chromosomes needs same gene expression levels as male

– “Inactivated” (genes prevented from being expressed) by heterochromatin

– Xist gene• Untranslated RNA

– Doesn’t code for proteins• Role in X-inactivation

Background

Image compliments of York Marahrens

Image compliments of York Marahrens

Background

Assaying Replication Timing

Data• Images of mouse cells

– Embryonic primary fibroblasts– Different cell types

• XaWT XiWT, XaWT Xi1lox, Xa1loxXiWT, Xa1lox Xi1lox

• DNA Fluorescence In Situ Hybridization (FISH)– Probes bind preferentially to X-chromosomes– Shows green in images

• BrdU immunostaining– shows red in images

Cell Cycle

BrdU Assay for

Replication

Timing

G1 S G2 M

3.5 h

6.0 h

Xi

Approach

DAPI Labeled Chromosomes

Xa

Xi

DAPI

X Paint Imaging

X Paint used to identify x chromosomes

BrdU Imaging

Samples pulsed with BrdU late in S-phase

Analysis

Automatic Quantification of BrdU incorporation in X-chromosomes

Overview

• Automatic detection of:– X-chromosomes in an image– Size (in pixels) of X-chromosomes– Amount of incorporated BrdU in X-

chromosome• # of red pixels

• Previous methods:– Manual outlining X-chromosome– Manual recording of quantities– Prone to error– Time consuming

Analysis

• Identify X-chromosomes using green intensities of image

Analysis

• Remove noise keeping only chromosomes

Analysis

• Remove noise keeping only chromosomes

Analysis

• Detected regions and original chromosomes not same size

Analysis

• Detected regions and original chromosomes not same size

Analysis

• Attempt to “grow” chromosomal region

Analysis

• Attempt to “grow” chromosomal region

Analysis

• Determine # total pixels in chromosomal region

Analysis

• Determine # red pixels in chromosomal region– Regions where BrdU present

Analysis

• Calculate the % of each X-chromosome that is replicated late– Presumed heterochromatic

• Perform analysis on entire image set

• Compare %’s between different types of cells

Conclusion

• Developed a pipeline to automatically quantify late replication in the X-chromosome using– BrdU immunostained/DNA FISH images– Computer graphics– Machine vision methods.

• Provides a solution orders of magnitude faster than manual methods

• Results will further studies in the regulation of chromatin structure of the X-chromosome– Xist Gene

• Carolyn Brown et al., (1991), Nature 349, 38-44.• Marahrens et al., (1998), Cell 92, p657-664.

Future Work

• Detect late replicating regions along the chromosome and correlate to sequence characteristics– Segment chromosomes

• Quantify BrdU values for various regions of chromosome

– Repetitive sequence environment• What type of repetitive sequences exist in a

particular segment of a chromosome

• Create Graphical User Interface (GUI)• Publish?

Acknowledgements

• SoCalBSI

• Dr. York Marahrens

• Dr. Moira Regelson

• Dr. Silvia Diaz-Perez

Questions?

• Epigenetics– The study of heritable changes in gene function that occur

without a change in the DNA sequence

• Histone– Any of several small, basic proteins most commonly found in

association with the DNA in the chromatin of eukaryotes

• Heterochromatin– Condensed chromosomal region– Thought to be genetically inert– Contain many repetitive sequences– Replicates late in the cell cycle

• Nucleosome– unit of chromatin consisting of a short length of DNA (about 140

bp) wrapped twice around a core of eight histone proteins

Acetylation is thought to directly facilitate transcription by loosening histone-DNA contacts

Methylation of some histones are correlated with transcriptional repression

Image compliments of York Marahrens

BackgroundPost Translational Modifications

Background

• Chromatin Classification– Euchromatin

• Genes can be expressed• Replicates earlier in S-phase

– Heterochromatin• Dense• Genes are suppressed• Late S-phase replication

The Cell Cycle

Background - Chromatin

• Structure comprised of Nucleosomes– Unit of chromatin

consisting of a short length of DNA (about 140 bp) wrapped twice around a core of eight histone proteins

• Post-translational modifications of histone proteins affect chromatin density

• Heritable (Epigenetic) trait of genes

http://www.albany.edu/~achm110/nucleosome.html