1. All Wrapped Up Chromatin-Level Gene Regulation

2. Genes can be regulated by chromatin organization GENETIC EPIGENETIC Pierce, B. 2005. Genetics, a conceptal approach. 2nd Ed. WH Freeman.

3. Chromatin Packaging Chromatin is the combination of DNA and proteins found in eukaryotic chromosomes. Proteins in chromatin are: –histones: small, positively charged –non-histone proteins: transcription factors, enzymes Packaging of DNA with proteins leads to compacted structure, which facilitates fitting the DNA into the nucleus

4. Nucleosomes: the basic chromatin unit Nucleosomes are formed by winding about 200 base pairs of the DNA duplex around a core of histone proteins. The histone core is made of: –2 molecules histone H2A –2 molecules histone H2B –2 molecules histone H3 –2 molecules histone H4 Another histone, H1, binds outside the core.

5. Nucleosome organization http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980218.html

6. Higher orders of chromatin packing produce fibers with wider diameters 1.Nucleosome (11 nm) 2.Solenoid (30 nm) 3.Loops (300 nm) 4.Coiled loops (700 nm) 5.Metaphase chromosome (1400 nm) Pierce, B. 2005. Genetics, a conceptual approach. 2nd Ed. WH Freeman. 1 2 3 4 5

7. The DNA component of chromatin can be covalently modified DNA methylation of cytosines CH 3 Only certain cytosines can be methylated. Sequence context matters. –In animals, CG –In plants, CG and CNG

8. Core histones can be covalently modified Example: Histone acetylation N-ARTKQTARKSTGGKAPRKQLATKAARKSAP 9 14 23 18 H3 Acetylation H-N-CH 2 -CH 3 ––N–C–C–– (CH 2 ) 4 O H ––N–C–C–– (CH 2 ) 4 NH 3 + O H Lysine Acetylated Lysine Acetylation Acetylation causes histones to lose some of their positive charge. This causes them to bind less tightly to the negatively charged DNA backbone. Pierce, B. 2005. Genetics, a conceptual approach. 2nd Ed. WH Freeman.

9. Consequences of chromatin modificaton Histone modification can reduce the positive charge on the proteins, thus altering their attraction for negatively charged DNA and loosening chromatin packing. Modification of both histones and cytosines can provide recognition sites for binding of other regulatory proteins, which in turn can alter chromatin packing. Acetylation Pierce, B. 2005. Genetics, a conceptual approach. 2nd Ed. WH Freeman.

10. Relationship of chromatin organization and gene expression Transcriptional Activity Chromatin Configuration Histone Modification DNA Methylation On Active Expressed Open Loosely packed Accessible to transcription machinery AcetylatedLow Off Inactive Silent Closed Tightly packed Not accessible to transcription machinery DeacetylatedHigh

11. Chromatin-level regulation of gene expression is called “epigenetic” Altered chromatin structure Altered expression Potentially reversible Independent of DNA sequence changes Frequently developmentally regulated Mitotically and / or meiotically heritable

12. Example: X-inactivation in mammals In mammals, there are many genes on the X chromosome. Females have two X chromosomes and males have only one. To compensate for possible differences in gene expression, due to the differences in X- chromosome number in females and males, one of the X’s in females is transcriptionally silenced by tightly packing the chromatin of one X.

13. Example: X-inactivation in mammals X X X XX X X X X X X X X X X X X In cats, the gene for fur color is on the X. There are two alleles, for orange and black.

14. X-inactivation fits the criteria for epigenetic regulation It is an example of altered gene expression (silencing) due to altered (more tightly packed) chromatin structure, not change in DNA sequence. It is developmentally imposed early in embryo formation. The new chromatin configuration is mitotically heritable, in that daughter cells maintain the chromatin state of the cell from which they came. It is reversible; during meiosis, the inactive X is re- activated, so that gametes inherit fully active X chromosomes.

15. Non-reversible epigenetic change in gene expression: paramutation In corn, synthesis of purple anthocyanin pigments requires a gene called booster (b), which codes for a transcription factor that activates the enzyme-coding genes in the pigment pathway. A strong allele, called B-I, is expressed at high levels and activates the pathway very well to produce deep purple coloration. Another allele, called B’, is expressed at lower levels and leads to weaker coloration. B-I B’

16. B’/B-I X B-I/B-I B’/B’ X B-I/B-I Paramutation exhibits non-Mendelian inheritance all B’ Non-Mendelian pattern of inheritance Images courtesy of Vicki Chandler, Univ of Arizona

17. Molecular explanation for paramutation Allelic interaction. One allele (B’) transfers its low expression state to another allele (B-I), converting B-I to B’. The conversion is chromatin-based. –B’ has a more closed chromatin configuration than B-I. –After association with B’, B-I assumes a closed chromatin configuration and is expressed at low levels like B’. The B-I-to-B’ conversion is permanent. B’ never reverts to B-I.

18. Chemically-induced transgenerational alterations in sexual function Many chemicals are related to estrogens and can disrupt endocrine function. Developing fetuses are especially sensitive to these so-called endocrine disrupting chemicals (EDCs). Treatment of pregnant mothers with EDCs can lead to changes in sexual development in their male offspring. The males can then transmit the changes to their offspring.

19. Chemically-induced defect can be passed to offspring pregnant mouse males; low sperm count X normal female males; low sperm count X normal female males; low sperm count X normal female males; low sperm count transient exposure to EDC F1 F2 F3 F4 Reference: Science (2005) 308:1466

20. Chemically-induced change is epigenetic Analysis of genes in sperm of the defective male mice revealed changes in DNA methylation of several genes. This altered pattern of DNA methylation was seen in mice from multiple generations (F1, F2, F3). Interpretation is that EDC induced a DNA methylation change in the germ line of male mice, and this change can be transmitted to offspring.

21. What we don’t know about chromatin-level regulation How many genes are targets for this level of regulation? How are those genes targeted? How are the chromatin modifications accomplished? What determines whether chromatin changes will be transient or permanent?

22. Summary DNA is packed into the nucleus by association with histone and non-histone proteins to form chromatin. Both the DNA and histone components of chromatin can be modified. Modifications change the configuration of chromatin and this in turn alters accessibility of genes for transcription. Some chromatin modifications can have transgenerational effects on gene expression.