Heterochromatin distribution and function in interphase Grant Farr (Freitag Lab) Neurospora crassa image: N. B. Raju, Stanford University

Long-term objectives To find fungus-specific inhibitors to better combat fungal infections in humans, animals and plants. To find fungus-specific inhibitors to better combat fungal infections in humans, animals and plants. To determine if centromere and heterochromatin organization are involved in polarized growth. To determine if centromere and heterochromatin organization are involved in polarized growth. X-Ray by ADAMS Health Care Center Aspergillosis

Histone H1-GFP tagged nuclei Vegetative cycle M. Springer Neurospora crassa

Polarized hyphal growth Histone H1-GFP tagged nuclei

Chromatin is heterogeneous Wolffe (1998) Chromatin euchromatin decondensed, active, DNA unmethylated histones hyperacetylated heterochromatin condensed, inactive, DNA methylated histones hypoacetylated Arabidopsis fission yeast mouse

Nuclear architecture HP1-GFP Histone H1-GFP Region of heterochromatin that may be associated with the telomeres Chromocenter

Role of heterochromatin in polarized growth HP1 mutants exhibit slow linear growth

Centromere-specific proteins CENP-A (CenH3) Centromere-specific histone H3 Other proteins: ~30 proteins (CENP-B to CENP-S) in humans ~60 known proteins in yeast Neurospora shares four identifiable proteins with humans: CenH3Cenp-ICenp-S CAC-3 (CAFp46/48) Young-Tae Chang and Young-Soo Kim from NYU department of Chemistry

Experimental outline 5’3’ 2. Digest plasmid and insert pGF1 XbaIBamHI 3. Ligate pMF Transformation into competent E.coli DH5  4. Purify DNA, linearize 5. Transform N. crassa 6. Analysis by epifluorescent microscopy 1. PCR

Amplification of genes for centromere-specific proteins +all three genes were amplified successfully + Cenp-S was fused to GFP + Cenp-I was fused to RFP + CAC-3 fusions did not work CENP- S CENP-ICAC-3 M 0.5 kb 1.0 kb 2.0 kb 3.0 kb Cenp-I CCG1 promoter RFP C-Terminus GFP CCG1 promoter Cenp-S C-Terminus

Transformation of Neurospora crassa Transformed linearized plasmid DNA carrying Cenp-S and Cenp-I fusion genes into two N. crassa strains each Genes targeted to the his-3 locus Initial transformations of Cenp-S Initial transformation of CENP-I

Backcross to purify strains Crosses: Cenp-S-GFP XN2557 (rid his-3 mat a) Cenp-S-GFPXN2556 (his-3 mat a; hpo RIP2 ) RFP-Cenp-IXN2557 (rid his-3 mat a) RFP-Cenp-IXN2556 (his-3 mat a; hpo RIP2 ) A.J.F. Griffiths, U.B.C. Uncrossed CENP-I RFP Crossed CENP-I with expected localization and less background noise

Cenp-S labels the centromere The HP1 cells previously characterized have similar localization. The HP1 cells previously characterized have similar localization. CenH3 localization is identical to Cenp-S localization. CenH3 localization is identical to Cenp-S localization. Cenp-S is identified as a part of the centromeric Cenp-A complex. Cenp-S is identified as a part of the centromeric Cenp-A complex. HP1 labeled with centromere and telomeric regions fluorescing Cenp-S-GFP

Imaging of the Labeled Centromeres Cenp-S gfp localized at the centromere

Centromeric localization of Cenp-S

Imaging of the Labeled Centromeres Cenp-I localized in the centromeric DNA region.

Confirming centromeric localization Hypha of different strains can form heterokaryons. Fusing SON-1-GFP and HP1-GFP strains with the new Cenp-S and Cenp-I strains will show simultaneous localization of the nuclear membrane and centromeres. Patrick Hickey (University of Edinburgh)

Cenp-I and HP1 co-localize partially RFP-CENP-I HP1-GFP

SON-1-GFP + RFP-CENP-I Cenp-I localization is centromeric SON-1 gfpCENP-I rfp

Is HP1 required for centromere localization? Hypothesis: Hypothesis: Heterochromatin is required for centromere localization. I tested this with the hpo X Cenp-S and Cenp-I crosses. I tested this with the hpo X Cenp-S and Cenp-I crosses.

Localization of CENP-S and CENP-I is maintained in HP1 mutants The same appears to be true for CenH3 (data not shown) Centromere localization appears independent of heterochromatin. RFP-CENP-I; hpo CENP-S-GFP; hpo

Possible Cenp-I mutant One of the Cenp-I X wildtype crosses gave us an hpo mutant like growth phenotype. One of the Cenp-I X wildtype crosses gave us an hpo mutant like growth phenotype. The phenotypes are separable by microscopy. The phenotypes are separable by microscopy. In the future we will sequence the Cenp-I genes to search for point mutations introduced by RIP in the cross. In the future we will sequence the Cenp-I genes to search for point mutations introduced by RIP in the cross.

Summary The Cenp-I and Cenp-S fusion proteins were expressed and appear to be located at centromeres. The Cenp-I and Cenp-S fusion proteins were expressed and appear to be located at centromeres. Lack of heterochromatin binding protein does not affect the localization of either proteins. Lack of heterochromatin binding protein does not affect the localization of either proteins. Possible mutant of Cenp-I affects apical growth. Possible mutant of Cenp-I affects apical growth.

Future Studies Construct an RFP-Cenp-S strain and a Cenp-I-GFP strains to better understand localization of the two using heterokaryons. Construct an RFP-Cenp-S strain and a Cenp-I-GFP strains to better understand localization of the two using heterokaryons. Slow-growing strains will be tested for the hpo mutation by DNA sequencing to verify that Cenp-S and Cenp-I localization were independent of HP1. Slow-growing strains will be tested for the hpo mutation by DNA sequencing to verify that Cenp-S and Cenp-I localization were independent of HP1. Use protein affinity tags (TAP, Myc, HA) to characterize other proteins in centromere complexes. Use protein affinity tags (TAP, Myc, HA) to characterize other proteins in centromere complexes. Find mutants in the Neurospora “knockout” collection that affect centromere localization and polarized growth. Find mutants in the Neurospora “knockout” collection that affect centromere localization and polarized growth.

Acknowledgements Howard Hughes Medical Institute Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Kevin Ahern Dr. Michael Freitag Dr. Michael Freitag Thomas Lew Thomas Lew