SU(HW) Fine-Tunes Nuclear Lamina Interactions Figure 4. SU(HW) alone is not essential for demarcation of LAD borders. Genome - NL interaction maps after knockdown and overexpression of SU(HW). (A) Western blot analysis of SU(HW) expression levels after knockdown (ctrl: control RNAi; kd1 and kd2: SU(HW) RNAi with two independent dsRNA fragments) and after overexpression (ctrl: control vector oe: overexression by transfection of SU(HW) under an Act5C promoter). 1st lane in each panel: transfected with Dam-LAM, 2nd lane with Dam-only. (B–C) Median NL interaction (log2 Dam-LAM/Dam ratio) across all aligned LAD borders (824 borders, 1st panel); border regions with SU(HW) present (220 borders, 2nd panel, red triangle represents SU(HW) at the borders), borders without SUH(HW) present (604 borders, 3rd panel). (B) Knockdown of SU(HW) (blue line) and control knockdown (grey line). (C) Overexpression of SU(HW) (orange line), and corresponding control (grey line). doi:10.1371/journal.pone.0015013.g004 the NL seems to be essential since the enhancer blocking activity of the gypsy insulator is disturbed by a lamin mutation [29]. Our data demonstrate that SU(HW) has an inhibitory effect on genome – NL interactions. This seems at odds with previous observations that the gypsy transposable element, which harbors a short array of SU(HW) binding motifs, can target flanking DNA to the nuclear periphery in a SU(HW) – dependent manner [43]. Possibly, the effect of SU(HW) on the nuclear location of gypsy is different from that on most LADs, perhaps due to a different sequence context. It is also possible that unknown factors cause SU(HW) to switch from an antagonist to an agonist of LAD – NL interactions depending on the cell type. At present, it is not known how LADs associate with the NL. Still, we can envision several mechanisms for the antagonistic action of SU(HW). For example, SU(HW) could form bulky DNA- associated complexes that locally disrupt NL interactions, which would be supported by the believe that SU(HW) forms aggregates at the nuclear periphery [42]. In addition distant gypsy elements PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e15013