1. Automatic Analysis of BrdU Incorporation for Replication Timing Assays Intern: Albert F. Cervantes Advisors: York Marahrens Moira Regelson Silvia Diaz-Perez

2. Overview Motivation Background The Project Future Work

3. 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

4. Background Image compliments of York Marahrens

5. Background

6. Assaying Replication Timing

7. Data Images of mouse cells –Embryonic primary fibroblasts –Different cell types Xa WT Xi WT, Xa WT Xi 1lox, Xa 1lox Xi WT, Xa 1lox Xi 1lox DNA Fluorescence In Situ Hybridization (FISH) –Probes bind preferentially to X- chromosomes –Shows green in images BrdU immunostaining –shows red in images

8. Cell Cycle

9. BrdU Assay for Replication Timing G1SG2M 3.5 h 6.0 h Xi

10. Approach DAPI Labeled Chromosomes Xa Xi DAPI

11. X Paint Imaging X Paint used to identify x chromosomes

12. BrdU Imaging Samples pulsed with BrdU late in S-phase

13. Analysis Automatic Quantification of BrdU incorporation in X-chromosomes

14. 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

15. Analysis Identify X-chromosomes using green intensities of image

16. Analysis Remove noise keeping only chromosomes

17. Analysis Remove noise keeping only chromosomes

18. Analysis Detected regions and original chromosomes not same size

19. Analysis Detected regions and original chromosomes not same size

20. Analysis Attempt to “grow” chromosomal region

21. Analysis Attempt to “grow” chromosomal region

22. Analysis Determine # total pixels in chromosomal region

23. Analysis Determine # red pixels in chromosomal region –Regions where BrdU present

24. 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

25. 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.

26. 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?

27. Acknowledgements SoCalBSI Dr. York Marahrens Dr. Moira Regelson Dr. Silvia Diaz-Perez

28. Questions?

29. 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

30. 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 Background Post Translational Modifications

31. Background Chromatin Classification –Euchromatin Genes can be expressed Replicates earlier in S-phase –Heterochromatin Dense Genes are suppressed Late S-phase replication

32. The Cell Cycle

33. 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