Oligonucleotides used in this study.

<div><p>In the malaria parasite <i>Plasmodium falciparum,</i> the expression of many genes is regulated by heterochromatin (HC) based on the histone mark tri-methylation of histone H3 lysine 9 (H3K9me3). HC assembly involves three distinct steps: <i>de novo</i> nucleation, spreading and maintenance. Nucleation, which consists in formation of HC in a previously euchromatic region, determines the specific genomic locations where HC occurs. This process is not well understood in malaria parasites. Here we investigated the DNA sequence <i>cis</i> determinants of HC nucleation in <i>P. falciparum</i>, using a screening approach based on integration of fragments from different heterochromatic genes into an euchromatic locus, followed by H3K9me3 chromatin immunoprecipitation (ChIP) analysis. We found that fragments of <i>var</i> gene upstream regions nucleated HC efficiently, whereas fragments from the <i>pfap2-g</i> upstream region or from the <i>mspdbl2</i> locus did not nucleate HC. Fragments from the beginning of the coding sequence (CDS) of <i>pfap2-g</i> nucleated HC with low efficiency, as evidenced by nucleation requiring long fragments of ~2 kb and occurring only in a fraction of the parasites. These results demonstrate that the primary DNA sequence is a main determinant of HC nucleation in <i>P. falciparum</i>. We also studied HC maintenance at the <i>pfap2-g</i> locus, which demonstrated that specific parts of the upstream region, different from the regions competent for HC nucleation, are required for maintenance. Together, our results provide initial insight into how HC is directed to specific loci and maintained in <i>P. falciparum</i>.</p></div>